Neurturin receptor

ABSTRACT

NTNRα, NTNRα extracellular domain (ECD), NTNRα variants, chimeric NTNRα (e.g., NTNRα immunoadhesion), and antibodies which bind thereto (including agonist and neutralizing antibodies) are disclosed. Various uses for these molecules are described, including methods to modulate cell activity and survival by response to NTNRα-ligands, for example NTN, by providing NTNRα to the cell.

BACKGROUND OF THE INVENTION

[0001] This is a non-provisional application filed under 37 CFR 1.53(b),claiming priority under USC Section 119(e) to provisional ApplicationSer. No. 60/063,258 filed on Oct. 24, 1997, provisional Application Ser.No. 60/049,818 filed on Jun. 9, 1997 and provisional Application Ser.No. 60/038,839 filed Feb. 18, 1997.

INTRODUCTION

[0002] 1. Technical Field

[0003] The present invention relates to a Neurturin (“NTN”) receptordesignated NTNRα (also referred to as GFRα2), and provides forNTNRα-encoding nucleic acid and amino acid sequences. In particular, theinvention relates to native sequence NTNRα, NTNRα variants, solubleNTNRα variants including NTNRα extracellular domain, chimeric NTNRα, andantibodies which bind to the NTNRα (including agonist and neutralizingantibodies), as well as various uses for these molecules. It alsorelates to assay systems for detecting ligands to NTNRα, systems forstudying the physiological role of NTN, diagnostic techniques foridentifying NTN-related conditions, therapeutic techniques for thetreatment of NTN-related and NTNRα-related conditions, and methods foridentifying molecules homologous to NTNRα.

[0004] 2. Background

[0005] Neurotrophic factors such as insulin-like growth factors, nervegrowth factor, brain-derived neurotrophic factor, neurotrophin-3, -4/5and -6, ciliary neurotrophic factor, GDNF, and neurturin have beenproposed as potential means for enhancing specific neuronal cellsurvival, for example, as a treatment for neurodegenerative diseasessuch as amyotrophic lateral sclerosis, Alzheimer's disease, stroke,epilepsy, Huntington's disease, Parkinson's disease, and peripheralneuropathy. It would be desirable to provide additional therapy for thispurpose. Protein neurotrophic factors, or neurotrophins, which influencegrowth and development of the vertebrate nervous system, are believed toplay an important role in promoting the differentiation, survival, andfunction of diverse groups of neurons in the brain and periphery.Neurotrophic factors are believed to have important signaling functionsin neural tissues, based in part upon the precedent established withnerve growth factor (NGF). NGF supports the survival of sympathetic,sensory, and basal forebrain neurons both in vitro and in vivo.Administration of exogenous NGF rescues neurons from cell death duringdevelopment. Conversely, removal or sequestration of endogenous NGF byadministration of anti-NGF antibodies promotes such cell death (Heumann,J. Exp. Biol., 132:133-150 (1987); Hefti, J. Neurosci., 6:2155-2162(1986); Thoenen et al., Annu. Rev. Physiol., 60:284-335 (1980)).

[0006] Additional neurotrophic factors related to NGF have since beenidentified. These include brain-derived neurotrophic factor (BDNF)(Leibrock, et al., Nature, 341:149-152 (1989)), neurotrophin-3 (NT-3)(Kaisho, et al., FEBS Lett., 266:187 (1990); Maisonpierre, et al.,Science, 247:1446 (1990); Rosenthal, et al., Neuron, 4:767 (1990)), andneurotrophin 4/5 (NT4/5) (Berkmeier, et al., Neuron, 7:857-866 (1991)).

[0007] Neurotrophins, similar to other polypeptide growth factors,affect their target cells through interactions with cell surfacereceptors. According to current understanding, two kinds oftransmembrane glycoproteins act as receptors for the knownneurotrophins. Equilibrium binding studies have shown thatneurotrophin-responsive neuronal cells possess a common low molecularweight (65,000 -80,000 Daltons), a low affinity receptor typicallyreferred to as p75^(LNGFR) or p75, and a high molecular weight(130,000-150,000 Dalton) receptor. The high affinity receptors aremembers of the trk family of receptor tyrosine kinases.

[0008] Receptor tyrosine kinases are known to serve as receptors for avariety of protein factors that promote cellular proliferation,differentiation, and survival. In addition to the trk receptors,examples of other receptor tyrosine kinases include the receptors forepidermal growth factor (EGF), fibroblast growth factor (FGF), andplatelet-derived growth factor (PDGF). Typically, these receptors spanthe cell membrane, with one portion of the receptor being intracellularand in contact with the cytoplasm, and another portion of the receptorbeing extracellular. Binding of a ligand to the extracellular portion ofthe receptor induces tyrosine kinase activity in the intracellularportion of the receptor, with ensuing phosphorylation of variousintracellular proteins involved in cellular signaling pathways.

[0009] Glial cell line-derived neurotrophic factor (“GDNF”) andNeurturin (“NTN”) are two, recently identified, structurally related,potent survival factors for sympathetic sensory and central nervoussystem neurons (Lin et al. Science 260:1130-1132 (1993); Henderson etal. Science 266:1062-1064 (1994); Buj-Bello et al., Neuron 15:821-828(1995); Kotzbauer et al. Nature 384:467-470 (1996)). Recently, GDNF wasshown to mediate its actions through a multi-component receptor systemcomposed of a ligand binding glycosyl-phosphatidyl inositol (GPI) linkedprotein (designated GDNFRα; also designated GFR-alpha-1) and thetransmembrane receptor tyrosine kinase Ret (Treanor et al. Nature382:80-83 (1996); Jing et al. Cell 85:1113-1124 (1996); Trupp et al.Nature 381:785-789 (1996); Durbec et al. Nature 381:789-793 (1996)). Themechanism by which the NTN signal is transmitted has not beenelucidated.

[0010] Aberrant expression of receptor tyrosine kinases (“RTK”)correlates with transforming ability. For example, carcinomas of theliver, lung, breast and colon show elevated expression of Eph RTK.Unlike many other tyrosine kinases, this elevated expression can occurin the absence of gene amplification or rearrangement. Moreover, Hek, ahuman RTK, has been identified as a leukemia-specific marker present onthe surface of a pre-B cell leukemia cell line. As with Eph, Hek alsowas overexpressed in the absence of gene amplification or rearrangementsin, for example, hemopoietic tumors and lymphoid tumor cell lines.Over-expression of Myk-1 (a murine homolog of human Htk (Bennett et al.,J. Biol. Chem., 269(19):14211-8 (1994)) was found in theundifferentiated and invasive mammary tumors of transgenic miceexpressing the Ha-ras oncogene. (Andres et al.,Oncogene, 9(5):1461-7(1994) and Andres et al., Oncogene, 9(8):2431 (1994)). Ret, the productof the c-ret proto-oncogene, is a member of the receptor tyrosine kinasesuperfamily.

[0011] In addition to their roles in carcinogenesis, a number oftransmembrane tyrosine kinases have been reported to play key rolesduring development. Some receptor tyrosine kinases are developmentallyregulated and predominantly expressed in embryonic tissues. Examplesinclude Cek1, which belongs to the FGF subclass, and the Cek4 and Cek5tyrosine kinases (Pasquale et al., Proc. Natl. Acad. Sci., USA,86:5449-5453 (1989); Sajjadi et al., New Biol., 3(8):769-78 (1991); andPasquale, Cell Regulation, 2:523-534 (1991)). Eph family members areexpressed in many different adult tissues, with several family membersexpressed in the nervous system or specifically in neurons (Maisonpierreet al., Oncogene, 8:3277-3288 (1993); Lai et al., Neuron, 6:691-704(1991)).

[0012] The aberrant expression or uncontrolled regulation of any one ofthese receptor tyrosine kinases can result in different malignancies andpathological disorders. Therefore, there exists a need to identify meansto regulate, control and manipulate receptor tyrosine kinases (“RTK”)and their associated ligands or GPI-inked receptors, in order to providenew and additional means for the diagnosis and therapy of receptortyrosine kinase pathway-related disorders and cellular processes. Thepresent application provides the clinician and researcher with suchmeans by providing new molecules that are specific for interacting withcertain RTK receptors. These compounds and their methods of use, asprovided herein, allow exquisite therapeutic control and specificity.Accordingly, it is an object of the present invention to provide animproved therapy for the prevention and/or treatment of neurologicalconditions and other conditions in which certain neurotrophic signalingpathways play a role.

[0013] These and other objects of the invention will be apparent to theordinarily skilled artisan upon consideration of the specification as awhole.

SUMMARY

[0014] A NTN receptor termed NTNRα, a soluble form of the receptor, anda NTNRα extracellular domain (“ECD”) are disclosed herein. Alsodisclosed are NTNRα polypeptides, optionally conjugated with or fused tomolecules which increase the serum half-lives thereof, and optionallyformulated as pharmaceutical compositions with a physiologicallyacceptable carrier.

[0015] Soluble NTNRa, including chimeric NTNRA molecules such as NTNRαECD immunoadhesins (having long serum half-lives) and epitope-taggedNTNRα ECD, that retain both ligand binding, preferably NTN binding, andreceptor signaling function (via Ret receptor tyrosine kinase) can beused to impart, restore, or enhance NTNRα-ligand (preferably NTN)responsiveness to cells. This responsiveness includes ligand-binding,Ret tyrosine phosphorylation and Ret-mediated downstream activity, whichcan result in modulation of cell activity such as survival or growth.The embodiments find use in vivo, in vitro or ex vivo. Soluble NTNRαforms that bind NTN but fail to interact with and activate Ret can beused as an antagonist to NTN ligand (by binding and sequestering NTN) toreduce activation of endogenous NTNRα. This is useful in conditionscharacterized by excess levels of NTN ligand and/or excess NTNRAactivation in a mammal. Bispecific immunoadhesins (for example,combining a NTNRα-ligand binding activity with a ligand-binding domainof another cytokine or neurotrophic factor receptor) can form highaffinity binding complexes for NTNRα-ligands and another factors,providing either antagonist activity (when Ret-activating function isabsent) or a means to enhance the attached ligands delivery.

[0016] Pharmaceutical compositions of soluble NTNRα, preferably ECD, canoptionally further include an NTNRα ligand, preferably NTN. Suchcompositions are useful where it is desirable to prolong the half-lifeof the ligand, provide slow, sustained release of ligand, impartNTNRα-ligand responsiveness to a target cell, and/or activate or enhanceendogenous cellular NTNRα or Ret activity directly. Optionally, thecomposition further contains one or more cytokines, neurotrophicfactors, or their agonist antibodies.

[0017] Also provided are methods for identifying a molecule which bindsto and/or activates NTNRα. Thus assays are provided to screen for oridentify NTNRα-ligand molecules (such as peptides, antibodies, and smallmolecules) that are agonists or antagonists of NTNRα. Such methodsgenerally involve exposing an immobilized NTNRα to a molecule suspectedof binding thereto and determining binding of the molecule to theimmobilized NTNRα and/or evaluating whether or not the moleculeactivates (or blocks activation of) the NTNRα. In order to identify suchNTN ligands, the NTNRα can be expressed on the surface of a cell andused to screen libraries of synthetic candidate compounds ornaturally-occurring compounds (e.g., from endogenous sources such asserum or cells). NTNRα can also be used as a diagnostic tool formeasuring serum levels of endogenous or exogenous NTNRα-ligand.

[0018] In a further embodiment, a method for purifying an NTNRα-ligandis provided. This finds use in commercial production and purification oftherapeutically active molecules that bind to this receptor. In oneembodiment the molecule of interest (generally in a compositioncomprising one or more contaminants) is adsorbed to immobilized NTNRα(e.g., NTNRα immunoadhesin immobilized on a protein A resin). Thecontaminants, by virtue of their inability to bind to the NTNRa, willgenerally not bind the resin. Accordingly, it is then possible torecover the molecule of interest from the resin by changing the elutionconditions, such that the ligand molecule is released from theimmobilized receptor.

[0019] Antibodies are provided that specifically bind to NTNRα.Preferred antibodies are monoclonal antibodies that are non-immunogenicin a human and bind to an epitope in the extracellular domain of thereceptor. Preferred antibodies bind the NTNRα with an affinity of atleast about 10⁶ L/mole, more preferably 10⁷ L/mole. Preferred antibodiesare agonist antibodies.

[0020] Antibodies, which bind to NTNRα, can be optionally fused to aheterologous polypeptide. The antibody or fusion finds particular use toisolate and purify NTNRα from a source of the receptor.

[0021] In a further aspect is provided a method for detecting NTNRα invitro or in vivo which includes the steps of contacting an NTNRαantibody with a sample suspected of containing the receptor, anddetecting if binding has occurred.

[0022] For certain applications it is desirable to have an agonistantibody. Such agonist antibodies are useful for activating NTNRα asdescribed for NTNRα-ligands such as NTN. Furthermore, these antibodiesare useful to treat conditions in which an effective amount of NTNRαactivation leads to a therapeutic benefit in the mammal. For example,the agonist antibody can be used to elicit an NTN response in a cellcomprising NTNRα and, preferably, Ret. For therapeutic applications itis desirable to prepare a composition having the agonist antibody and aphysiologically acceptable carrier. Optionally, the composition furthercontains one or more cytokines, neurotrophic factors, or their agonistantibodies.

[0023] In other embodiments, the antibody is a neutralizing antibody.Such molecules can be used to treat conditions characterized by unwantedor excessive activation of NTNRα.

[0024] In addition to the above, the invention provides isolated nucleicacid molecules, expression vectors and host cells encoding NTNRα whichcan be used in the recombinant production of NTNRα as described herein.The isolated nucleic acid molecules and vectors are also useful toprepare transgenic animals, for gene therapy applications to treatpatients with NTNRα defects or increase responsiveness of cells to NTNRαligands, or alternatively to decrease NTNRα activity (as by use ofantisense nucleic acid).

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIGS. 1A-1B depict the nucleic acid sequence (SEQ ID NO.: 1) ofthe sense strand of the cDNA encoding full length human NTNRα, thehNTNRα-encoding sequence (SEQ ID NO.: 2), and the deduced amino acidsequence of full length hNTNRα (SEQ ID NO.: 3). Nucleotides are numberedat the beginning of the sense strand. Amino acid residues are numberedat the beginning of the amino acid sequence.

[0026] FIGS. 2A-2B depict the nucleic acid sequence (SEQ ID NO.: 4) ofthe sense strand of the cDNA encoding full length rat NTNRα, therNTNRα-encoding sequence (SEQ ID NO.: 5), and the deduced amino acidsequence of full length rNTNRα (SEQ ID NO.: 6). Nucleotides are numberedat the beginning of the sense strand. Amino acid residues are numberedat the beginning of the amino acid sequence.

[0027] FIGS. 3A-3B compare hNTNRα-, rNTNRα-, and rGDNFRα-encodingnucleic acids.

[0028]FIG. 4 is a comparison of the hNTNRα, rNTNRα, and rGDNFRαproteins, with features indicated. Signal peptides are indicated by asolid line. Signal cleavage sites are marked with arrows. Potentialglycosylation sites are shaded. The hydrophobic domain of the GPIattachment site is doubly underlined. The small amino acid residues thatconstitute a cleavage/attachment site for GPI-linked proteins are markedwith asterisks. Consensus cysteine residues are indicated by a solidcircle. The extracellular domain (“ECD”) is flanked by the signalpeptide and the GPI-attachment site.

[0029]FIG. 5 is a comparison of the amino acid sequences of hNTNRα andhGDNFRα.

[0030] FIGS. 6A-6D depict binding of I¹²⁵ NTN and GDNF to NTNRα- orGDNFRα-expressing cells and displacement by unlabeled NTN. FIGS. 6A and6C show the binding of 125I mouse NTN (¹²⁵I-mNTN) to rat GDNFRα(“rGDNFRα”) or rat NTNRα (“rNTNRα”), respectively. FIGS. 6B and 6D showthe binding of ¹²⁵IrGDNF (¹²⁵I-rGDNF) to rat GDNFRα (“rGDNFRα”) or ratNTNRα (“rNTNRα”), respectively. As depicted by the Scatchard analysis,displayed in the inset of FIG. 6B, GDNF binds GDNFRα with a K_(d) valueof 3 pM. A similar K_(d) was reported in a cell based assay (Jing et al.Cell 85:1113-1124 (1996)). Mouse NTN binds rNTNRα with a K_(d) value of10 pM (see inset to FIG. 6C). Human NTNRα displayed a similar bindingspecificity as rat NTNRα (data not shown). Although in these experimentsno binding of ¹²⁵I NTN to GDNFRα (FIG. 6A) and of ¹²⁵IrGDNF to NTNRα(FIG. 6D) were detected, experiments performed with biotinylated NTN andGDNF revealed low affinity binding (K_(d) above 1 mM) of NTN to GDNFRα,and vice versa.

[0031] FIGS. 7A-7F depict interaction between NTN, NTNRα and Ret. FIG.7A depicts binding of ¹²⁵I NTN to cells expressing NTNRα. Consistentwith the prediction that NTNRα is a GPI-linked protein, binding of ¹²⁵INTN to NTNRα expressing cells was reduced by 50-70% following treatmentwith PIPLC. FIG. 7B depicts survival response of embryonic, rat spinalmotoneurons to GDNF or NTN. In agreement with its receptor distribution,NTN is a potent survival factor for spinal motoneurons. FIG. 7C depictssurvival response of embryonic, rat spinal motoneurons to NTN or BDNF inthe presence of PIPLC and a soluble NTNRα. PIPLC treatment reduced thesurvival response to NTN by 50-90% without changing the response toBDNF. Soluble NTNRα (sRα) restores the response of PIPLC-treatedmotoneurons to NTN. FIG. 7D depicts NTN induction of tyrosinephosphorylation of Ret in neuroblastoma TGW-1 cells. FIG. 7E depicts NTNinduction of phosphorylation of ERK in TGW-1. FIG. 7F depict theNTN-responsiveness (e.g., Ret phosphorylation) imparted by anNTN-soluble NTNRα complex to Ret-expressing cells. Legends:(Con)=untransfected cells. (Ret)=cells transfected with Ret alone.(Ra+Ret)=cells transfected with Ret and NTNRα. In all cases, cells wereexposed to NTN (100 ng/ml) and then processed for immunoprecipitationwith NTN antisera.

[0032]FIGS. 8A to 8C depict the survival response of dopaminergicneurons to soluble Ret-activating forms of NTNRα and GDNFRαextracellular domains.

[0033]FIG. 9 depicts the ratio of DOPAC to dopamine (expressed as theinjected side as a percentage of the uninjected side, mean±sem) invarious brain regions, particularly the striatum, from rats injected inone striatum with NTN, a soluble form of NTNRα, both NTN and NTNRα, orvehicle.

DETAILED DESCRIPTION

[0034] In describing the present invention, the following terms will beemployed and are intended to be defined as indicated below.

[0035] The terms “NTNRα” (also designated GFR-alpha-2) or “NTNRαpolypeptide” when used herein encompass native sequence NTNRα; NTNRαvariants; NTNRα extracellular domain; and chimeric NTNRα (each of whichis defined herein). Optionally, the NTNRα is not associated with nativeglycosylation. “Native glycosylation” refers to the carbohydratemoieties which are covalently attached to NTNRα when it is produced inthe mammalian cell from which it is derived in nature. Accordingly,human NTNRα produced in a non-human cell is an example of a NTNRα whichmay “not be associated with native glycosylation.” Sometimes, the NTNRαis unglycosylated (e.g.,as a result of being produced recombinantly in aprokaryote).

[0036] A “native sequence NTNRα” comprises a polypeptide having the sameamino acid sequence as a NTNRα derived from nature. Thus, a nativesequence NTNRα can have the amino acid sequence of naturally occurringrat NTNRα, murine NTNRα, human NTNRα, or NTNRα from any other mammalianspecies. Such native sequence NTNRα polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence NTNRα” specifically encompasses naturally-occurringtruncated forms of the NTNRα, naturally-occurring variant forms(e.g.,alternatively spliced forms), and naturally-occurring allelicvariants of the NTNRα. The preferred native sequence NTNRα is a maturenative sequence NTNRα. NTNRα sequence for human and rat are shown inFIGS. 1A-1D and 2A-2B. Preferred molecules are those comprising anucleic acid molecule that is capable of hybridizing under moderate, andmore preferably under stringent hybridization conditions, with the DNAsequence encoding the human NTN receptor shown in FIGS. 1A-1B. In oneembodiment the NTNRα nucleic acid hybridizes at 42° C. in 20% formamidewith the DNA sequence encoding the NTN receptor shown in FIGS. 1A-1B. Inanother embodiment a nucleic acid molecule is capable of hybridizing at42° C. in 20% formamide with a DNA sequence of at least 10 contiguousbases, and preferably at least 20 contiguous bases, more preferably withat least 45 bases, and even more preferably with at least 60 basesencoding a portion of the complete NTN receptor shown in FIGS. 1A-1B or2A-2B. Preferred sequences do not hybridize GDNFRα sequences undersimilar conditions.

[0037] The “NTNRα extracellular domain” (ECD) is a form of the NTNRαwhich is essentially free of the transmembrane and cytoplasmic domainsof NTNRα, i.e., has less than 1% of such domains, preferably 0.5 to 0%of such domains, and more preferably 0.1 to 0% of such domains.Ordinarily, the NTNRα ECD will have an amino acid sequence having atleast about 60% amino acid sequence identity with the amino acidsequence of the ECD of an NTNRα, for example as indicated in FIGS. 1A-1Bor 2A-2B for NTNRα or the corresponding sequences provided herein, e.g.mouse sequences, preferably at least about 65%, more preferably at leastabout 75%, even more preferably at least about 80%, even more preferablyat least about 90%, with increasing preference of 95%, to at least 99%amino acid sequence identity, and finally to 100% identity, and thusincludes NTNRα variants as defined below. Preferred sequences will be atleast 16 amino acids long, preferably at least 20 amino acids long, andeven more preferably at least 40 amino acids long.

[0038] “NTNRα variant” means a biologically active NTNRα as definedbelow having less than 100% sequence identity (but at least 60%identity) with a NTNRα, for example, having the deduced amino acidsequence shown in FIGS. 1A-1B or 2A-2B for NTNRα or with the sequencesprovided herein. Such NTNRα variants include NTNRα polypeptides whereinone or more amino acid residues are added at the N- or C-terminus of, orwithin, a NTNRα sequence; from about one to thirty amino acid residuesare deleted, and optionally substituted by one or more amino acidresidues; and derivatives of the above polypeptides, wherein an aminoacid residue has been covalently modified so that the resulting producthas a non-naturally occurring amino acid. Ordinarily, a biologicallyactive NTNRα variant will have an amino acid sequence having about 60%amino acid sequence identity with the amino acid sequence of anaturally-occurring NTNRα (e.g., as shown in FIGS. 1A-1B or 2A-2B or thecorresponding sequences provided herein), preferably at least about 65%,more preferably at least about 75%, even more preferably at least about80%, even more preferably at least about 90%, with increasing preferenceof 95%, to at least 99% amino acid sequence identity, and finally to100% identity.

[0039] A “chimeric NTNRα” is a polypeptide comprising full-length NTNRαor one or more domains thereof (e.g.,the extracellular domain) fused orbonded to heterologous polypeptide. The chimeric NTNRα will generallyshare at least one biological property in common with NTNRα. Examples ofchimeric NTNRαs include immunoadhesins and epitope-tagged NTNRα.

[0040] The term “immunoadhesin” is used interchangeably with theexpression “NTNRA-immunoglobulin chimera” and refers to a chimericmolecule that combines a portion of the NTNRα (generally theextracellular domain thereof) with an immunoglobulin sequence. Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG1, IgG2, IgG3or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferably IgG1 or IgG3.

[0041] The term “epitope-tagged” when used herein refers to a chimericpolypeptide comprising NTNRα fused to a “tag polypeptide”. The tagpolypeptide has enough residues to provide an epitope against which anantibody thereagainst can be made, yet is short enough such that it doesnot interfere with biological activity of the NTNRα. The tag polypeptidepreferably also is fairly unique so that the antibody thereagainst doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8-50 amino acid residues (preferably between about 9-30residues). Preferred are poly-histidine sequences, which bind nickel,allowing isolation of the tagged protein by Ni-NTA chromatography asdescribed (Lindsay et al. Neuron 17:571-574 (1996)), for example.

[0042] “Isolated NTNRα” means NTNRα that has been purified from a NTNRαsource or has been prepared by recombinant or synthetic methods and issufficiently free of other peptides or proteins (1) to obtain at least15 and preferably 20 amino acid residues of the N-terminal or of aninternal amino acid sequence by using a spinning cup sequenator or thebest commercially available amino acid sequenator marketed or asmodified by published methods as of the filing date of this application,or (2) to homogeneity by SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins.

[0043] “Essentially pure” protein means a composition comprising atleast about 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight. “Essentiallyhomogeneous” protein means a composition comprising at least about 99%by weight of protein, based on total weight of the composition.

[0044] “Biological property” when used in conjunction with either“NTNRα” or “isolated NTNRα” means having an effector or antigenicfunction or activity that is directly or indirectly caused or performedby native sequence NTNRα (whether in its native or denaturedconformation). Effector functions include ligand binding, andenhancement of survival, differentiation and/or proliferation of cells(especially proliferation of cells). However, effector functions do notinclude possession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against native sequence NTNRα.

[0045] An “antigenic function” means possession of an epitope orantigenic site that is capable of cross-reacting with antibodies raisedagainst native sequence NTNRα. The principal antigenic function of aNTNRα polypeptide is that it binds with an affinity of at least about10⁶ L/mole to an antibody raised against native sequence NTNRα.Ordinarily, the polypeptide binds with an affinity of at least about 10⁷L/mole. The antibodies used to define “antigenic function” are rabbitpolyclonal antibodies raised by formulating the NTNRα in Freund'scomplete adjuvant, subcutaneously injecting the formulation, andboosting the immune response by intraperitoneal injection of theformulation until the titer of the anti-NTNRα antibody plateaus.

[0046] “Biologically active” when used in conjunction with either“NTNRα” or “isolated NTNRα” means a NTNRα polypeptide that exhibits orshares an effector function of native sequence NTNRα and that may (butneed not), in addition, possess an antigenic function. A principaleffector function of the NTNRα is its ability to bind NTN. Anotherprincipal effector function of NTNRα is activating Ret tyrosine kinase(resulting in Ret autophosphorylation) to activate downstream pathwaysmediated by Ret signaling function.

[0047] “Antigenically active” NTNRα is defined as a polypeptide thatpossesses an antigenic function of NTNRα and that may (but need not) inaddition possess an effector function.

[0048] “Percent amino acid sequence identity” with respect to the NTNRαsequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues in the NTNRαsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the candidate NTNRα sequence shall beconstrued as affecting sequence identity or homology.

[0049] “NTN ligand” is a molecule which binds to and preferablyactivates native sequence NTNRα. The ability of a molecule to bind toNTNRα can be determined, for example, by the ability of the putativeligand to bind to NTNRα immunoadhesin coated on an assay plate, forexample. Specificity of binding can be determined by comparing bindingto GDNFRα. Competitive binding of NTN to NTNRα is a preferred propertyof the ligand. The thymidine incorporation assay provides another meansfor screening for ligands which activate NTNRα function.

[0050] A “thymidine incorporation assay” can be used to screen formolecules which activate the NTNRα. In order to perform this assay, IL-3dependent Baf3 cells (Palacios et al., Cell, 41:727-734 (1985)) arestably transfected with full length native sequence NTNRα as describedherein and Ret. The NTNRα/Ret/Baf3 cells so generated are starved ofIL-3 for 24 hours in a humidified incubator at 37° C. in 5%CO₂ and air.Following IL-3 starvation, the cells are plated out in 96 well culturedishes with, or without, a test sample containing a potential agonist(such test samples are optionally diluted) and cultured for 24 hours ina cell culture incubator. 20 μl of serum free RPMI media containing 1μCi of ³H thymidine is added to each well for the last 6-8 hours. Thecells are then harvested in 96 well filter plates and washed with water.The filters are then counted using a Packard Top Count MicroplateScintillation Counter, for example. Agonists are expected to induce astatistically significant increase (to a P value of 0.05) in ³H uptake,relative to control. Preferred agonists leads to an increase in ³Huptake which is at least two fold of that of the control. Other assaysare described herein.

[0051] An “isolated” NTNRα nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the NTNRα nucleic acid. An isolated NTNRα nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated NTNRα nucleic acid molecules therefore aredistinguished from the NTNRα nucleic acid molecule as it exists innatural cells. However, an isolated NTNRα nucleic acid molecule includesNTNRα nucleic acid molecules contained in cells that ordinarily expressNTNRα where, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

[0052] The expression “control sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and possibly, other as yet poorlyunderstood sequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0053] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0054] As used herein, the expressions “cell,” “cell line,” and “cellculture” are used interchangeably and all such designations includeprogeny. Thus, the words “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

[0055] The term “antibody” is used in the broadest sense andspecifically covers monoclonal antibodies, antibody compositions withpolyepitopic specificity, bispecific antibodies, diabodies, andsingle-chain molecules, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv), so long as they exhibit the desired biologicalactivity.

[0056] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256: 495 (1975), or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567(Cabilly et al.)). The “monoclonal antibodies” may also be isolated fromphage antibody libraries using the techniques described in Clackson etal., 624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

[0057] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0058] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a Primatized™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

[0059] “Non-immunogenic in a human” means that upon contacting thepolypeptide of interest in a physiologically acceptable carrier and in atherapeutically effective amount with the appropriate tissue of a human,no state of sensitivity or resistance to the polypeptide of interest isdemonstrable upon the second administration of the polypeptide ofinterest after an appropriate latent period (e.g., 8 to 14 days).

[0060] By “agonist antibody” is meant an antibody which is a NTNRαligand, able to activate native sequence NTNRα.

[0061] A “neutralizing antibody” is one which is able to block orsignificantly reduce an effector function of native sequence NTNRα. Forexample, a neutralizing antibody may inhibit or reduce NTNRα activationby a NTN ligand, as determined, for example, in a neurite survivalassays, a NTN binding assay, or other assays taught herein or known inthe art.

[0062] The phrase “enhancing proliferation of a cell” encompasses thestep of increasing the extent of growth and/or reproduction of the cellrelative to an untreated cell either in vitro or in vivo. An increase incell proliferation in cell culture can be detected by counting thenumber of cells before and after exposure to a molecule of interest. Theextent of proliferation can be quantified via microscopic examination ofthe degree of confluence. Cell proliferation can also be quantifiedusing the thymidine incorporation assay described herein.

[0063] By “enhancing differentiation of a cell” is meant the act ofincreasing the extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e. cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g.,identifying morphologicalchanges in the cell).

[0064] “Physiologically acceptable” carriers, excipients, or stabilizersare ones which are nontoxic to the cell or mammal being exposed theretoat the dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or polyethylene glycol (PEG).

[0065] As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1,IgG2, IgG3, and IgG4) that is responsible for increasing the in vivoserum half-life of the IgG molecule. Exemplary salvage receptor bindingepitope sequences include HQNLSDGK; HQNISDGK; HQSLGTQ; VISSHLGQ; andPKNSSMISNTP.

[0066] The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); neurotrophic factors or nerve growth factors suchas NGF-β, NT-3, NT4, NT-6, BDNF, CNTF, GDNF, AL-1 and other eph-receptorfamily ligands; platelet-growth factor; transforming growth factors(TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines. Also included are genetically engineeredmolecules with cytokine activity such as TrkA-IgG or other solublereceptor chimeras.

[0067] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented.

[0068] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal is human.

[0069] By “solid phase” is meant a non-aqueous matrix to which a reagentof interest (e.g.,the NTNRα or an antibody thereto) can adhere. Examplesof solid phases encompassed herein include those formed partially orentirely of glass (e.g.,controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g.,an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

[0070] Modes for carrying out the invention are presented herein. Glialcell line-derived neurotrophic factor (“GDNF”) and Neurturin (“NTN”) aretwo structurally related, potent survival factors for sympatheticsensory and central nervous system neurons (Lin et al. Science260:1130-1132 (1993); Henderson et al. Science 266:1062-1064 (1994);Buj-Bello et al., Neuron 15:821-828 (1995); Kotzbauer et al. Nature384:467-470 (1996)). Whereas GDNF was shown to mediate its actionsthrough a multi-component receptor system composed of a ligand bindingglycosyl-phosphatidyl inositol (GPI) linked protein (designated GDNFRA)and the transmembrane tyrosine kinase Ret (Treanor et al. Nature382:80-83 (1996); Jing et al. Cell 85:1113-1124 (1996); Trupp et al.Nature 381:785-789 (1996); Durbec et al. Nature 381:789-793 (1996)), themechanism by which the NTN signal is transmitted has not been previouslyelucidated. Described herein is the isolation, sequence, and tissuedistribution of a GPI-linked protein and its gene, designated NTNRα,which is shown to modulate response to NTN but not GDNF. It is shownherein that it is structurally related to GDNFRα. Using recombinantproteins in a cell free system, it is shown that NTNRα binds NTN (Kd˜10pM) but not GDNF, and that NTN does not bind GDNFRα with a highaffinity. Also shown is that cellular responses to NTN require thepresence of NTNRα. Ligand bound NTNRα induces phosphorylation of thetyrosine kinase receptor Ret. These findings identify Ret and NTNRα,respectively, as signaling and ligand binding components of a receptorfor NTN and related ligands. This defines a novel neurotrophic anddifferentiation factor receptor family of receptors containing a sharedtransmembrane protein tyrosine kinase (Ret) and a ligand specificGPI-linked protein (NTNRα).

[0071] Glial cell line-derived neurotrophic factor (“GDNF”) (Lin et al.,Science, 260:1130-1132 (1993); WO 93/06116, which are incorporatedherein in its entirety), is a potent survival factor for midbraindopaminergic (Lin et al., Science, 260:1130-1132 (1993); Strömberg etal., Exp. Neurol., 124:401412 (1993); Beck et al., Nature, 373:339-341(1995); Kearns et al., Brain Res., 672:104-111 (1995); Tomac et al.,Nature, 373:335-339 (1995)) spinal motor (Henderson et al., Science,266:1062-1064 (1994);Oppenheim et al., Nature, 373:344-346 (1995); Yanet al., Nature, 373:341-344 (1995)) and noradrenergic neurons (Arenas etal., Neuron, 15:1465-1473 (1995)), which degenerate in Parkinson'sdisease (Hirsch et al., Nature, 334:345-348 (1988); Hornykiewicz Mt.Sinai J. Med., 55:11-20 (1988)), amyotrophic lateral sclerosis (Hirano,Amyotrophic Lateral Sclerosis and Other Motor Neuron Diseases, P.Rowland, ed. (New York: Raven Press, Inc.) pp. 91-101 (1991)), andAlzheimer's disease (Marcynuik et al., J. Neurol. Sci., 76:335-345(1986); Cash et al., Neurology, 37:4246 (1987); Chan-Palay et al., Comp.Neurol., 287:373-392 (1989)) respectively. Based on mice geneticallyengineered to lack GDNF, additional biological roles for GDNF have beenreported: the development and/or survival of enteric, sympathetic, andsensory neurons and the renal system, but not for catecholaminergicneurons in the central nervous system (CNS) (Moore et al. Nature382:76-79 (1996); Pichel et al. Nature 382:73-76 (1996); Sanchez et al.Nature 382:70-73 (1996)). Despite the physiological and clinicalimportance of GDNF, little is known about its mechanism of action.

[0072] Cytokine receptors frequently assemble into multi-subunitcomplexes. Sometimes, the α subunit of this complex is involved inbinding the cognate growth factor and the β-subunit may contain anability to transduce a signal to the cell. Without wishing to be boundby theory, these receptors have been assigned to three subfamiliesdepending on the complexes formed. Subfamily 1 includes the receptorsfor EPO, granulocyte colony-stimulating factor (G-CSF), interleukin-4(IL-4), interleukin-7 (IL-7), growth hormone (GH), and prolactin (PRL).Ligand binding to receptors belonging to this subfamily is thought toresult in homodimerization of the receptor. Subfamily 2 includesreceptors for IL-3, granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin-5 (IL-5), interleukin-6 (IL-6), leukemiainhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotrophicfactor (CNTF). Subfamily 2 receptors are heterodimers having ana-subunit for ligand binding, and β-subunit (either the shared β-subunitof the IL-3, GM-CSF, and IL-5 receptors or the gp130 subunit of theIL-6, LIF, OSM, and CNTF receptors) for signal transduction. Subfamily 3contains only the interleukin-2 (IL-2) receptor. The β and γ subunits ofthe IL-2 receptor complex are cytokine-receptor polypeptides whichassociate with the α-subunit of the unrelated Tac antigen.

[0073] The present invention is based on the discovery of the NTNRα, aprotein that binds NTN with a high affinity. The experiments describedherein demonstrate that this molecule is a receptor which appears toplay a role in mediating responses to NTN. In particular, this receptorhas been found to be present in a variety of tissue and cellpopulations, including neurons, thus indicating that NTN ligands, suchas agonist antibodies, can be used to stimulate proliferation, growth,survival, differentiation, metabolism, or regeneration of NTNRα- andRet-containing cells.

[0074] Techniques suitable for the production of NTNRα are well known inthe art and include isolating NTNRα from an endogenous source of thepolypeptide, peptide synthesis (using a peptide synthesizer) andrecombinant techniques (or any combination of these techniques). Thepreferred technique for production of NTNRα is a recombinant techniqueto be described below.

[0075] Most of the discussion below pertains to recombinant productionof NTNRα by culturing cells transformed with a vector containing NTNRαnucleic acid and recovering the polypeptide from the cell culture. It isfurther envisioned that the NTNRα of this invention may be produced byhomologous recombination, as provided for in WO 91/06667, published May16, 1991.

[0076] Briefly, this method involves transforming primary human cellscontaining a NTNRα-encoding gene with a construct (ie., vector)comprising an amplifiable gene (such as dihydrofolate reductase (DHFR)or others discussed below) and at least one flanking region of a lengthof at least about 150 bp that is homologous with a DNA sequence at thelocus of the coding region of the NTNRα gene to provide amplification ofthe NTNRα gene. The amplifiable gene must be at a site that does notinterfere with expression of the NTNRα gene. The transformation isconducted such that the construct becomes homologously integrated intothe genome of the primary cells to define an amplifiable region.

[0077] Primary cells comprising the construct are then selected for bymeans of the amplifiable gene or other marker present in the construct.The presence of the marker gene establishes the presence and integrationof the construct into the host genome. No further selection of theprimary cells need be made, since selection will be made in the secondhost. If desired, the occurrence of the homologous recombination eventcan be determined by employing PCR and either sequencing the resultingamplified DNA sequences or determining the appropriate length of the PCRfragment when DNA from correct homologous integrants is present andexpanding only those cells containing such fragments. Also if desired,the selected cells may be amplified at this point by stressing the cellswith the appropriate amplifying agent (such as methotrexate if theamplifiable gene is DHFR), so that multiple copies of the target geneare obtained. Preferably, however, the amplification step is notconducted until after the second transformation described below.

[0078] After the selection step, DNA portions of the genome,sufficiently large to include the entire amplifiable region, areisolated from the selected primary cells. Secondary mammalian expressionhost cells are then transformed with these genomic DNA portions andcloned, and clones are selected that contain the amplifiable region. Theamplifiable region is then amplified by means of an amplifying agent ifnot already amplified in the primary cells. Finally, the secondaryexpression host cells now comprising multiple copies of the amplifiableregion containing NTNRα are grown so as to express the gene and producethe protein.

[0079] The conserved structure and sequence of the mammalian NTNRα andthe elucidation of the cDNA sequence which encodes the rat and mousereceptor, as well as human sequences disclosed herein, make it possibleto clone gene sequences from other mammals which encode the NTNRα. Ofparticular interest to the present invention is the ability to clone thehuman NTNRα molecules using the sequences disclosed herein. The DNAencoding NTNRα may be obtained from any cDNA library prepared fromtissue believed to possess the NTNRα mRNA and to express it at adetectable level, as shown herein in the Examples. Accordingly, NTNRαDNA can be conveniently obtained from a cDNA library prepared, forexample, from mammalian fetal liver, brain, muscle, intestine, andperipheral nerves. The NTNRα-encoding gene may also be obtained from agenomic library or by oligonucleotide synthesis.

[0080] Libraries are screened with probes (such as antibodies to theNTNRα or oligonucleotides of about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures as described in chapters 10-12 of Sambrook et al., MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1989). An alternative means to isolate the gene encoding NTNRα isto use PCR methodology as described in section 14 of Sambrook et al.,supra.

[0081] A preferred method of practicing this invention is to usecarefully selected oligonucleotide sequences to screen cDNA librariesfrom various human tissues, preferably human fetal liver. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.Preferred sequences are obtained from the naturally-occurring NTNRαdisclosed herein.

[0082] The oligonucleotide must be labeled such that it can be detectedupon hybridization to DNA in the library being screened. The preferredmethod of labeling is to use ³²P-labeled ATP with polynucleotide kinase,as is well known in the art, to radiolabel the oligonucleotide. However,other methods may be used to label the oligonucleotide, including, butnot limited to, biotinylation or enzyme labeling.

[0083] Amino acid sequence variants of NTNRα are prepared by introducingappropriate nucleotide changes into the NTNRα DNA, or by synthesis ofthe desired NTNRα polypeptide. Such variants represent insertions,substitutions, and/or specified deletions of, residues within or at oneor both of the ends of the amino acid sequence of a naturally occurringNTNRα, such as the NTNRα shown in FIGS. 1A-1B or 2A-2B or sequencesdisclosed herein. Preferably, these variants represent insertions and/orsubstitutions within or at one or both ends of the mature sequence,and/or insertions, substitutions and/or specified deletions within or atone or both of the ends of the signal sequence of the NTNRα. Anycombination of insertion, substitution, and/or specified deletion ismade to arrive at the final construct, provided that the final constructpossesses the desired biological activity as defined herein. The aminoacid changes also may alter post-translational processes of the NTNRα,such as changing the number or position of glycosylation sites, alteringthe membrane anchoring characteristics, and/or altering theintracellular location of the NTNRα by inserting, deleting, or otherwiseaffecting the leader sequence of the NTNRα.

[0084] Variations in the native sequence as described above can be madeusing any of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Theseinclude oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. See also, for example, Table I thereinand the discussion surrounding this table for guidance on selectingamino acids to change, add, or delete.

[0085] The nucleic acid (e.g., cDNA or genomic DNA) encoding the NTNRαis inserted into a replicable vector for further cloning (amplificationof the DNA) or for expression. Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

[0086] The NTNRαs of this invention may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. In general, the signal sequence may be a component ofthe vector, or it may be a part of the NTNRα DNA that is inserted intothe vector. The heterologous signal sequence selected preferably is onethat is recognized and processed (ie., cleaved by a signal peptidase) bythe host cell. For prokaryotic host cells that do not recognize andprocess the native NTNRα signal sequence, the signal sequence issubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase, 1pp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,α factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182 issued Apr. 23,1991), or acid phosphatase leader, the C. albicans glucoamylase leader(EP 362,179 published Apr. 4, 1990), or the signal described in WO90/13646 published Nov. 15, 1990. In mammalian cell expression thenative signal sequence (e.g., the NTNRα presequence that normallydirects secretion of NTNRα from human cells in vivo) is satisfactory,although other mammalian signal sequences may be suitable, such assignal sequences from other animal NTNRαs, and signal sequences fromsecreted polypeptides of the same or related species, as well as viralsecretory leaders, for example, the herpes simplex gD signal.

[0087] The DNA for such precursor region is ligated in reading frame toDNA encoding the mature NTNRα or a soluble variant thereof.

[0088] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

[0089] Most expression vectors are “shuttle” vectors, i.e., they arecapable of replication in at least one class of organisms but can betransfected into another organism for expression. For example, a vectoris cloned in E. coli and then the same vector is transfected into yeastor mammalian cells for expression even though it is not capable ofreplicating independently of the host cell chromosome.

[0090] DNA may also be amplified by insertion into the host genome. Thisis readily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of NTNRα DNA. However, the recovery of genomic DNA encodingNTNRα is more complex than that of an exogenously replicated vectorbecause restriction enzyme digestion is required to excise the NTNRαDNA.

[0091] Expression and cloning vectors should contain a selection gene,also termed a selectable marker. This gene encodes a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

[0092] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene produce a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin, mycophenolic acid and hygromycin.

[0093] Another example of suitable selectable markers for mammaliancells are those that enable the identification of cells competent totake up the NTNRα nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesNTNRα. Amplification is the process by which genes in greater demand forthe production of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of NTNRα are synthesized from the amplified DNA.Other examples of amplifiable genes include metallothionein-I and -II,preferably primate metallothionein genes, adenosine deaminase, ornithinedecarboxylase, etc. A preferred vector system is provided in U.S. Pat.No. 5,561,053.

[0094] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium that contains methotrexate (Mtx), a competitive antagonist ofDHFR. An appropriate host cell when wild-type DHFR is employed is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub et al., Proc. Natl. Acad.Sci. USA, 77:4216 (1980). The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingNTNRα. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060).

[0095] Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding NTNRα, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

[0096] A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39(1979)). The trpl gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of thetrpl lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

[0097] In addition, vectors derived from the 1.6 μm circular plasmidpKD1 can be used for transformation of Kluyveromyces yeasts. Bianchi etal., Curr. Genet., 12:185 (1987). More recently, an expression systemfor large-scale production of recombinant calf chymosin was reported forK. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., Bio/Technology, 9:968-975 (1991).

[0098] Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the NTNRαnucleic acid. Promoters are untranslated sequences located upstream (5′)to the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of particularnucleic acid sequence, such as the NTNRα nucleic acid sequence, to whichthey are operably linked. Such promoters typically fall into twoclasses, inducible and constitutive. Inducible promoters are promotersthat initiate increased levels of transcription from DNA under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters are operably linked toNTNRα-encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native NTNRα promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the NTNRα DNA. However, heterologous promoters arepreferred, as they generally permit greater transcription and higheryields of NTNRα as compared to the native NTNRα promoter.

[0099] Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275:615(1978); Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase, atryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057(1980); EP 36,776), and hybrid promoters such as the tac promoter.deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983). However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding NTNRα (Siebenlist et al., Cell, 20:269(1980)) using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also will contain aShine-Delgarno (S.D.) sequence operably linked to the DNA encodingNTNRα.

[0100] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

[0101] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et a., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0102] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

[0103] NTNRα transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, and from the promoter normallyassociated with the NTNRα sequence, provided such promoters arecompatible with the host cell systems.

[0104] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment that also contains the SV40viral origin of replication. Fiers et al., Nature, 273:113 (1978);Mulligan et al., Science, 209:1422-1427 (1980); Pavlakis et al., Proc.Natl. Acad. Sci. USA, 78:7398-7402 (1981). The immediate early promoterof the human cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment. Greenaway et al., Gene, 18:355-360 (1982). Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso Gray et al., Nature, 295:503-508 (1982) on expressing cDNA encodingimmune interferon in monkey cells; Reyes et al., Nature, 297:598-601(1982) on expression of human β-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus;Canaani et al., Proc. Natl. Acad. Sci. USA, 79:5166-5170 (1982) onexpression of the human interferon β1 gene in cultured mouse and rabbitcells; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781(1982) on expression of bacterial CAT sequences in CV-1 monkey kidneycells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLacells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

[0105] Transcription of a DNA encoding the NTNRα of this invention byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5′ (Laimins et al., Proc. Natl. Acad. Sci. USA, 78:993(1981)) and 3′ (Lusky et al., Mol. Cell Bio., 3:1108 (1983)) to thetranscription unit, within an intron (Banerji et al., Cell, 33:729(1983)), as well as within the coding sequence itself. Osborne et al.,Mol. Cell Bio., 4:1293 (1984). Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theNTNRα-encoding sequence, but is preferably located at a site 5′ from thepromoter.

[0106] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding NTNRα.

[0107] Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

[0108] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain 294 (ATCC 31,446) and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65:499 (1980).

[0109] Particularly useful in the practice of this invention areexpression vectors that provide for the transient expression inmammalian cells of DNA encoding NTNRα. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Sambrook et al.,supra, pp. 16.17 - 16.22. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive identification of polypeptides encoded by cloned DNAs, as wellas for the rapid screening of such polypeptides for desired biologicalor physiological properties. Thus, transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of NTNRα that are biologically active NTNRα.

[0110] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of NTNRα in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058. A particularlyuseful plasmid for mammalian cell culture expression of NTNRα is pRK5(EP 307,247) or pSVI6B. WO 91/08291 published Jun. 13, 1991.

[0111] Suitable host cells for cloning or expressing the DNA in thevectors herein are the prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published Apr. 12, 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC27,325) are suitable. These examples are illustrative rather thanlimiting. Strain W3110 is a particularly preferred host or parent hostbecause it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell should secrete minimal amountsof proteolytic enzymes. For example, strain W3110 may be modified toeffect a genetic mutation in the genes encoding proteins, with examplesof such hosts including E. coli W3110 strain 27C7. The complete genotypeof 27C7 is tonAΔptr3 phoAΔE15 Δ(argF-lac)169 ompTΔ degP41kan′. Strain27C7 was deposited on Oct. 30, 1991 in the American Type CultureCollection as ATCC No. 55,244. Alternatively, the strain of E. colihaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990 may be employed. Alternatively still, methods ofcloning, e.g., PCR or other nucleic acid polymerase reactions, aresuitable.

[0112] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forNTNRα-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe (Beach et al., Nature, 290:140 (1981); EP 139,383 published May 2,1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,supra) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourtet al., J. Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., supra), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278(1988)); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979));Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene,26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA,81:1470-1474 (1984)) and A. niger. Kelly et al., EMBO J., 4:475-479(1985).

[0113] Suitable host cells for the expression of glycosylated NTNRα arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6:47-55(1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature,315:592-594 (1985). A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

[0114] Plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the NTNRα-encoding DNA. During incubation of the plant cellculture with A. tumefaciens, the DNA encoding the NTNRα is transferredto the plant cell host such that it is transfected, and will, underappropriate conditions, express the NTNRα-encoding DNA. In addition,regulatory and signal sequences compatible with plant cells areavailable, such as the nopaline synthase promoter and polyadenylationsignal sequences. Depicker et al., J. Mol. Appl. Gen., 1:561(1982). Inaddition, DNA segments isolated from the upstream region of the T-DNA780 gene are capable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published Jun. 21, 1989.

[0115] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure. See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

[0116] Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for NTNRα production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

[0117] Transfection refers to the taking up of an expression vector by ahost cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CAPO₄ and electroporation. Successful transfectionis generally recognized when any indication of the operation of thisvector occurs within the host cell.

[0118] Transformation means introducing DNA into an organism so that theDNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described in section 1.82 ofSambrook et al., supra, or electroporation is generally used forprokaryotes or other cells that contain substantial cell-wall barriers.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published Jun. 29, 1989. In addition, plants may betransfected using ultrasound treatment as described in WO 91/00358published Jan. 10, 1991.

[0119] For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham et al., Virology, 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216 issuedAug. 16, 1983. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. USA, 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, etc., may also beused. For various techniques for transforming mammalian cells, see Keownet al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0120] Prokaryotic cells used to produce the NTNRα polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

[0121] The mammalian host cells used to produce the NTNRα of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham et al. Meth. Enz., 58:44 (1979),Barnes et al., Anal. Biochem.,102:255 (1980), U.S. Pat. Nos. 4,767,704;4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195;or U.S. Pat. Re. 30,985 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0122] In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

[0123] The host cells referred to in this disclosure encompass cells inculture as well as cells that are within a host animal.

[0124] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

[0125] Gene expression, alternatively, can be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75:734-738 (1980).

[0126] Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared as described herein.

[0127] NTNRα (e.g., NTNRα ECD) preferably is recovered from the culturemedium as a secreted polypeptide, although it also may be recovered fromhost cell lysates. If the NTNRα is membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g. Triton-X100).

[0128] When NTNRα is produced in a recombinant cell other than one ofhuman origin, the NTNRα is completely free of proteins or polypeptidesof human origin. However, it is necessary to purify NTNRα fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to NTNRα. As a first step, the culturemedium or lysate can be centrifuged to remove particulate cell debris.NTNRα can then be purified from contaminant soluble proteins andpolypeptides with the following procedures, which are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica; chromatofocusing; immunoaffinity; epitope-tag binding resin;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; and protein A Sepharose columns to removecontaminants such as IgG.

[0129] NTNRα variants in which residues have been deleted, inserted, orsubstituted are recovered in the same fashion as native sequence NTNRα,taking account of any substantial changes in properties occasioned bythe variation. Immunoaffinity resins, such as a monoclonal anti-NTNRαresin, can be employed to absorb the NTNRα variant by binding it to atleast one remaining epitope.

[0130] A protease inhibitor such as phenyl methyl sulfonyl fluoride(PMSF) also may be useful to inhibit proteolytic degradation duringpurification, and antibiotics may be included to prevent the growth ofadventitious contaminants.

[0131] Covalent modifications of NTNRα polypeptides are included withinthe scope of this invention. Both native sequence NTNRα and amino acidsequence variants of the NTNRα may be covalently modified. One type ofcovalent modification of the NTNRα is introduced into the molecule byreacting targeted amino acid residues of the NTNRα with an organicderivatizing agent that is capable of reacting the N-terminal residue,the C-terminal residue, or with selected side chains.

[0132] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0133] Histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1M sodium cacodylateat pH 6.0.

[0134] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

[0135] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed under alkaline conditionsbecause of the high pK_(a) of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas with the arginine epsilon-amino group.

[0136] The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method being suitable.

[0137] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R-N═C═N-R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

[0138] Derivatization with bifunctional agents is useful forcrosslinking NTNRα to a water-insoluble support matrix or surface foruse in the method for purifying anti-NTNRα antibodies, and viceversa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-((p-azidophenyl)dithio)propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0139] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

[0140] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terninal amine, and amidation of any C-terminalcarboxyl group.

[0141] Another type of covalent modification of the NTNRα polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. By altering is meantdeleting one or more carbohydrate moieties found in native NTNRα, and/oradding one or more glycosylation sites that are not present in thenative NTNRα.

[0142] Glycosylation of polypeptides is typically either N-linked or0-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

[0143] Addition of glycosylation sites to the NTNRα polypeptide isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the native NTNRα sequence (for O-linked glycosylationsites). For ease, the NTNRα amino acid sequence is preferably alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the NTNRα polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. The DNAmutation(s) may be made using methods described above and in U.S. Pat.No. 5,364,934, supra.

[0144] Another means of increasing the number of carbohydrate moietieson the NTNRα polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. These procedures are advantageous in thatthey do not require production of the polypeptide in a host cell thathas glycosylation capabilities for N- or O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO 87/05330 published Sep.11, 1987, and in Aplin et al., CRC Crit. Rev. Biochem., 259-306 (1981).

[0145] Removal of carbohydrate moieties present on the NTNRα polypeptidemay be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al., Arch. Biochem. Biophys., 259:52(1987) and by Edge et al., Anal.Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. EnzymoL,138:350 (1987).

[0146] Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al., J.Biol. Chem., 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

[0147] Another type of covalent modification of NTNRα comprises linkingthe NTNRα polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

[0148] Variants can be assayed as taught herein. A change in theimmunological character of the NTNRα molecule, such as affinity for agiven antibody, can be measured by a competitive-type immunoassay. Otherpotential modifications of protein or polypeptide properties such asredox or thermal stability, hydrophobicity, susceptibility toproteolytic degradation, or the tendency to aggregate with carriers orinto multimers are assayed by methods well known in the art.

[0149] This invention encompasses chimeric polypeptides comprising NTNRαfused to a heterologous polypeptide. A chimeric NTNRα is one type ofNTNRα variant as defined herein. In one preferred embodiment, thechimeric polypeptide comprises a fusion of the NTNRα with a tagpolypeptide which provides an epitope to which an anti-tag antibody ormolecule can selectively bind. The epitope-tag is generally provided atthe amino- or carboxyl- terminus of the NTNRA. Such epitope-tagged formsof the NTNRα are desirable, as the presence thereof can be detectedusing a labeled antibody against the tag polypeptide. Also, provision ofthe epitope tag enables the NTNRα to be readily purified by affinitypurification using the anti-tag antibody. Affinity purificationtechniques and diagnostic assays involving antibodies are describedlater herein.

[0150] Tag polypeptides and their respective antibodies are well knownin the art. Examples include the flu HA tag polypeptide and its antibody12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)); the c-myctag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan etal., Molecular and Cellular Biology, 5:3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody. Paborsky et al.,Protein Engineering, 3(6):547-553 (1990). Other tag polypeptides havebeen disclosed. Examples include the Flag-peptide (Hopp et al.,BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science, 255:192-194 (1992)); an α-tubulin epitope peptide (Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)); and the T7 gene 10protein peptide tag. Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990). Once the tag polypeptide has been selected, anantibody thereto can be generated using the techniques disclosed herein.A C-terminal poly-histidine sequence tag is preferred. Poly-histidinesequences allow isolation of the tagged protein by Ni-NTA chromatographyas described (Lindsay et al. Neuron 17:571-574 (1996)), for example.

[0151] The general methods suitable for the construction and productionof epitope-tagged NTNRα are the same as those disclosed hereinabove.NTNRα-tag polypeptide fusions are most conveniently constructed byfusing the cDNA sequence encoding the NTNRα portion in-frame to the tagpolypeptide DNA sequence and expressing the resultant DNA fusionconstruct in appropriate host cells. Ordinarily, when preparing theNTNRα-tag polypeptide chimeras of the present invention, nucleic acidencoding the NTNRα will be fused at its 3′ end to nucleic acid encodingthe N-terminus of the tag polypeptide, however 5′ fusions are alsopossible.

[0152] Epitope-tagged NTNRα can be conveniently purified by affinitychromatography using the anti-tag antibody. The matrix to which theaffinity antibody is attached is most often agarose, but other matricesare available (e.g. controlled pore glass orpoly(styrenedivinyl)benzene). The epitope-tagged NTNRα can be elutedfrom the affinity column by varying the buffer pH or ionic strength oradding chaotropic agents, for example.

[0153] Chimeras constructed from a receptor sequence linked to anappropriate immunoglobulin constant domain sequence (immunoadhesins) areknown in the art. Immunoadhesins reported in the literature includefusions of the T cell receptor (Gascoigne et al., Proc. Natl.Acad. Sci.USA, 84: 2936-2940 (1987)); CD4* (Capon et al., Nature 337: 525-531(1989); Traunecker et al., Nature, 339: 68-70 (1989); Zettmeissl et al.,DNA Cell BioL USA, 9: 347-353 (1990); Byrn et al., Nature, 344: 667-670(1990)); L-selectin (homing receptor) ((Watson et al., J. Cell. Biol.,110:2221-2229 (1990); Watson et al., Nature, 349: 164-167 (1991)); CD44*(Aruffo et al., Cell, 61: 1303-1313 (1990)); CD28* and B7* (Linsley etal., J. Exp. Med., 173: 721-730(1991)); CTLA-4 (Lisley etal., J. Exp.Med. 174: 561-569 (1991)); CD22* (Stamenkovic et al., Cell, 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol., 27: 2883-2886(1991); Peppel et al., J. Exp. Med., 174:1483-1489 (1991)); NP receptors(Bennett et al., J. Biol. Chem. 266:23060-23067 (1991)); and IgEreceptor α* (Ridgway et al., J. Cell. Biol., 1 15:abstr. 1448 (1991)),where the asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily.

[0154] The simplest and most straightforward immunoadhesin designcombines the binding region(s) of the “adhesin” protein with the hingeand Fc regions of an immunoglobulin heavy chain. Ordinarily, whenpreparing the NTNRα-immunoglobulin chimeras of the present invention,nucleic acid encoding the extracellular domain of the NTNRα will befused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

[0155] Typically, in such fusions the encoded chimeric polypeptide willretain at least functionally active hinge and CH2 and CH3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

[0156] The precise site at which the fusion is made is not critical;particular sites are well known and may be selected in order to optimizethe biological activity, secretion or binding characteristics of theNTNRα-immunoglobulin chimeras.

[0157] In some embodiments, the NTNRα-immunoglobulin chimeras areassembled as monomers, or hetero- or homo-multimer, and particularly asdimers or tetramers, essentially as illustrated in WO 91/08298.

[0158] In a preferred embodiment, the NTNRα extracellular domainsequence is fused to the N-terminus of the C-terminal portion of anantibody (in particular the Fc domain), containing the effectorfunctions of an immunoglobulin, e.g. immunoglobulin G₁ (IgG1). It ispossible to fuse the entire heavy chain constant region to the NTNRαextracellular domain sequence. However, more preferably, a sequencebeginning in the hinge region just upstream of the papain cleavage site(which defines IgG Fc chemically; residue 216, taking the first residueof heavy chain constant region to be 114, or analogous sites of otherimmunoglobulins) is used in the fusion. In a particularly preferredembodiment, the NTNRα amino acid sequence is fused to the hinge regionand CH2 and CH3, or to the CH1, hinge, CH2 and CH3 domains of an IgG1,IgG2, or IgG3 heavy chain. The precise site at which the fusion is madeis not critical, and the optimal site can be determined by routineexperimentation.

[0159] In some embodiments, the NTNRα-immunoglobulin chimeras areassembled as multimer, and particularly as homo-dimers or -tetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basic fourunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in multimeric form in serum. In the case ofmultimer, each four unit may be the same or different.

[0160] Alternatively, the NTNRα extracellular domain sequence can beinserted between immunoglobulin heavy chain and light chain sequencessuch that an immunoglobulin comprising a chimeric heavy chain isobtained. In this embodiment, the NTNRα sequence is fused to the 3′ endof an immunoglobulin heavy chain in each arm of an immunoglobulin,either between the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom et al.,Mol. Immunol., 28:1027-1037 (1991).

[0161] Although the presence of an immunoglobulin light chain is notrequired in the immunoadhesins of the present invention, animmunoglobulin light chain might be present either covalently associatedto an NTNRα-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the NTNRα extracellular domain. In the former case, DNAencoding an immunoglobulin light chain is typically coexpressed with theDNA encoding the NTNRa-immunoglobulin heavy chain fusion protein. Uponsecretion, the hybrid heavy chain and the light chain will be covalentlyassociated to provide an immunoglobulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodssuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

[0162] In a preferred embodiment, the immunoglobulin sequences used inthe construction of the immunoadhesins of the present invention are froman IgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG1 and IgG3 immunoglobulin sequencesis preferred. A major advantage of using IgG1 is that IgG1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG3 hingeis longer and more flexible, so it can accommodate larger adhesindomains that may not fold or function properly when fused to IgG1.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For NTNRα immunoadhesins designed for in vivoapplication, the pharmacokinetic properties and the effector functionsspecified by the Fc region are important as well. Although IgG1, IgG2and IgG4 all have in vivo half-lives of 21 days, their relativepotencies at activating the complement system are different. IgG4 doesnot activate complement, and IgG2 is significantly weaker at complementactivation than IgG1. Moreover, unlike IgG1, IgG2 does not bind to Fcreceptors on mononuclear cells or neutrophils. While IgG3 is optimal forcomplement activation, its in vivo half-life is approximately one thirdof the other IgG isotypes. Another important consideration forimmunoadhesins designed to be used as human therapeutics is the numberof allotypic variants of the particular isotype. In general, IgGisotypes with fewer serologically-defined allotypes are preferred. Forexample, IgG1 has only four serologically-defined allotypic sites, twoof which (G1m and 2) are located in the Fc region; and one of thesesites G1m1, is non-immunogenic. In contrast, there are 12serologically-defined allotypes in IgG3, all of which are in the Fcregion; only three of these sites (G3m5, 11 and 21) have one allotypewhich is nonimmunogenic. Thus, the potential immunogenicity of a γ3immunoadhesin is greater than that of a γ1 immunoadhesin.

[0163] With respect to the parental immunoglobulin, a useful joiningpoint is just upstream of the cysteines of the hinge that form thedisulfide bonds between the two heavy chains. In a frequently useddesign, the codon for the C-terminal residue of the NTNRα part of themolecule is placed directly upstream of the codons for the sequenceDKTHTCPPCP of the IgG1 hinge region.

[0164] The general methods suitable for the construction and expressionof immunoadhesins are the same as those disclosed hereinabove withregard to NTNRα. NTNRα immunoadhesins are most conveniently constructedby fusing the cDNA sequence encoding the NTNRα portion in-frame to an IgcDNA sequence. However, fusion to genomic Ig fragments can also be used(see, e.g., Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84:2936-2940(1987); Aruffo et al., Cell, 61:1303-1313 (1990); Stamenkovic et al.,Cell, 66:1133-1144 (1991)). The latter type of fusion requires thepresence of Ig regulatory sequences for expression. cDNAs encoding IgGheavy-chain constant regions can be isolated based on published sequencefrom cDNA libraries derived from spleen or peripheral blood lymphocytes,by hybridization or by polymerase chain reaction (PCR) techniques. ThecDNAs encoding the NTNRα and Ig parts of the immunoadhesin are insertedin tandem into a plasmid vector that directs efficient expression in thechosen host cells. For expression in mammalian cells, pRK5-based vectors(Schall et al., Cell, 61:361-370 (1990)) and CDM8-based vectors (Seed,Nature, 329:840 (1989)) can be used. The exact junction can be createdby removing the extra sequences between the designed junction codonsusing oligonucleotide-directed deletional mutagenesis (Zoller et al,Nucleic Acids Res., 10:6487 (1982); Capon et al., Nature, 337:525-531(1989)). Synthetic oligonucleotides can be used, in which each half iscomplementary to the sequence on either side of the desired junction;ideally, these are 36 to 48-mers. Alternatively, PCR techniques can beused to join the two parts of the molecule in-frame with an appropriatevector.

[0165] The choice of host cell line for the expression of NTNRαimmunoadhesins depends mainly on the expression vector. Anotherconsideration is the amount of protein that is required. Milligramquantities often can be produced by transient transfections. Forexample, the adenovirus EIA-transformed 293 human embryonic kidney cellline can be transfected transiently with pRK5-based vectors by amodification of the calcium phosphate method to allow efficientimmunoadhesin expression. CDM8-based vectors can be used to transfectCOS cells by the DEAE-dextran method (Aruffo et al., Cell, 61:1303-1313(1990); Zettmeissl et al., DNA Cell Biol. US, 9:347-353 (1990)). Iflarger amounts of protein are desired, the immunoadhesin can beexpressed after stable transfection of a host cell line. For example, apRK5-based vector can be introduced into Chinese hamster ovary (CHO)cells in the presence of an additional plasmid encoding dihydrofolatereductase (DHFR) and conferring resistance to G418. Clones resistant toG418 can be selected in culture; these clones are grown in the presenceof increasing levels of DHFR inhibitor methotrexate; clones areselected, in which the number of gene copies encoding the DHFR andimmunoadhesin sequences is co-amplified. If the immunoadhesin contains ahydrophobic leader sequence at its N-terminus, it is likely to beprocessed and secreted by the transfected cells. The expression ofimmunoadhesins with more complex structures may require uniquely suitedhost cells; for example, components such as light chain or J chain maybe provided by certain myeloma or hybridoma cell hosts (Gascoigne etal., 1987, supra, Martin et al., J. Virol., 67:3561-3568 (1993)).

[0166] Immunoadhesins can be conveniently purified by affinitychromatography. The suitability of protein A as an affinity liganddepends on the species and isotype of the immunoglobulin Fc domain thatis used in the chimera. Protein A can be used to purify immunoadhesinsthat are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol. Meth., 62:1-13 (1983)). Protein G is recommended for all mouseisotypes and for human γ3 (Guss et al., EMBO J., 5:1567-1575 (1986)).The matrix to which the affinity ligand is attached is most oftenagarose, but other matrices are available. Mechanically stable matricessuch as controlled pore glass or poly(styrenedivinyl)benzene allow forfaster flow rates and shorter processing times than can be achieved withagarose. The conditions for binding an immunoadhesin to the protein A orG affinity column are dictated entirely by the characteristics of the Fcdomain; that is, its species and isotype. Generally, when the properligand is chosen, efficient binding occurs directly from unconditionedculture fluid. One distinguishing feature of immunoadhesins is that, forhuman γ1 molecules, the binding capacity for protein A is somewhatdiminished relative to an antibody of the same Fc type. Boundimmunoadhesin can be efficiently eluted either at acidic pH (at or above3.0), or in a neutral pH buffer containing a mildly chaotropic salt.This affinity chromatography step can result in an immunoadhesinpreparation that is >95% pure.

[0167] Other methods known in the art can be used in place of, or inaddition to, affinity chromatography on protein A or G to purifyimmunoadhesins. Immunoadhesins behave similarly to antibodies inthiophilic gel chromatography (Hutchens et al., Anal. Biochem.,159:217-226 (1986)) and immobilized metal chelate chromatography(Al-Mashikhi et al., J. Dairy Sci., 71:1756-1763 (1988)). In contrast toantibodies, however, their behavior on ion exchange columns is dictatednot only by their isoelectric points, but also by a charge dipole thatmay exist in the molecules due to their chimeric nature.

[0168] If desired, the immunoadhesins can be made bispecific. Thus, theimmunoadhesins of the present invention may combine a NTNRαextracellular domain and a domain, such as the extracellular domain, ofanother cytokine or neurotrophic factor receptor subunit. Exemplarycytokine receptors from which such bispecific immunoadhesin moleculescan be made include TPO (or mpl ligand), EPO, G-CSF, IL-4, IL-7, GH,PRL, IL-3, GM-CSF, IL-5, IL-6, LIF, OSM,CNTF, GDNF and IL-2 receptors.For bispecific molecules, trimeric molecules, composed of a chimericantibody heavy chain in one arm and a chimeric antibody heavychain-light chain pair in the other arm of their antibody-like structureare advantageous, due to ease of purification. In contrast toantibody-producing quadromas traditionally used for the production ofbispecific immunoadhesins, which produce a mixture of ten tetramers,cells transfected with nucleic acid encoding the three chains of atrimeric immunoadhesin structure produce a mixture of only threemolecules, and purification of the desired product from this mixture iscorrespondingly easier.

[0169] The NTNRα protein and NTNRα gene are believed to find ex vivo orin vivo therapeutic use for administration to a mammal, particularlyhumans, in the treatment of diseases or disorders, related to neurturinactivity or benefited by neurturin-responsiveness. See Kotzbauer et al.Nature 384:467-470 (1996), which is specifically incorporated herein byreference. Conditions particularly amenable to treatment with theembodiments of the invention are those related to Ret expression or thatbenefit by Ret activation, particularly of the downstream pathwaysmediated by Ret. See Treanor et al. Nature 382:80-83 (1996); Jing et alCell 85:1113-1124 (1996); Trupp et al Nature 381:785-789 (1996); andDurbec et al. Nature 381:789-793 (1996), which are specificallyincorporated herein by reference. Particularly preferred are neurologicdisorders, preferably central nervous system disorders, disorders of thekidney, hematopoietic disorders related to the spleen, and entericnervous system disorders. The patient is administered an effectiveamount of NTNRα protein, peptide fragment, or variant of the invention.Therapeutic methods comprising administering NTNRα, NTNRα agonists (e.g.NTN), NTNRα antagonists (which compete with and bind endogenous NTN butfail to activate Ret), or anti-NTNRα antibodies are within the scope ofthe present invention. The present invention also provides forpharmaceutical compositions comprising NTNRα protein, peptide fragment,or derivative in a suitable pharmacologic carrier. The NTNRα protein,peptide fragment, or variant may be administered systemically orlocally. Applicable to the methods taught herein, the receptor proteincan be optionally administered prior to, after, or preferablyconcomitantly with (or in complex with) NTN or other NTNRα ligand. Astaught herein, NTNRα can be provided to target cells in the absence ofNTN to increase the responsiveness of those cells to subsequentlyadministered NTN or NTN agonist.

[0170] Certain conditions can benefit from an increase in NTN (or otherNTNRα-ligand) responsiveness. It may therefore be beneficial to increasethe number of or binding affinity of NTNRα in cells of patientssuffering from such conditions. This can be achieved throughadministration of soluble NTNRα, optionally complexed with NTNRα-ligand,preferably NTN, or by gene therapy using NTNRα-encoding nucleic acid.Selective expression of recombinant NTNRα in appropriate cells could beachieved using NTNRα genes controlled by tissue specific or induciblepromoters or by producing localized infection with replication defectiveviruses carrying a recombinant NTNRα gene. Conditions which may benefitfrom increased sensitivity to NTN include, but are not limited to,motoneuron disorders including amyotrophic lateral sclerosis,Werdnig-Hoffmann disease, chronic proximal spinal muscular atrophy, andGuillain-Barre syndrome. Additional conditions include those involvingsympathetic neurons, particularly where increased survival orNTN-responsiveness is desired. Conditions where increased survival orNTN-responsiveness of sensory neurons, including peripheral sensoryneurons, and central nervous system neurons, including dopaminergicneurons, is desirable are also suitably treated with embodiments of theinvention. Accordingly, treatment of neurological disorders associatedwith diabetes, Parkinson's disease, Alzheimer's disease, andHuntington's chorea are provided herein. NTN finds particular use intreatment of Parkinson's disease. The present methods can also beapplied to conditions related to non-neuronal cells that express NTNRα.In fact, since NTNRα serves to activate Ret, conditions associated withRet-expressing cells can be treated with the embodiments of theinvention.

[0171] A disease or medical disorder is considered to be nerve damage ifthe survival or function of nerve cells and/or their axonal processes iscompromised. Such nerve damage occurs as the result conditions including(a) Physical injury, which causes the degeneration of the axonalprocesses and/or nerve cell bodies near the site of the injury; (b)Ischemia, as a stroke; (c) Exposure to neurotoxins, such as the cancerand AIDS chemotherapeutic agents such as cisplatin and dideoxycytidine(ddC), respectively; (d) Chronic metabolic diseases, such as diabetes orrenal dysfunction; and (e) Neurodegenerative diseases such asParkinson's disease, Alzheimer's disease, and Amyotrophic LateralSclerosis (ALS), which cause the degeneration of specific neuronalpopulations. Conditions involving nerve damage include Parkinson'sdisease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, stroke,diabetic polyneuropathy, toxic neuropathy, and physical damage to thenervous system such as that caused by physical injury of the brain andspinal cord or crush or cut injuries to the arm and hand or other partsof the body, including temporary or permanent cessation of blood flow toparts of the nervous system, as in stroke.

[0172] The NTNRα gene is expressed in muscle cells and associatedneurons. Accordingly, the present invention provides for methods oftreating muscle cell disorders comprising administering to a patient inneed of such treatment the compounds of the invention. Muscle celldisorders which may benefit from such treatment include but are notlimited to the following progressive muscular dystrophies: Duchenne,Becker, Emery-Dreifuss, Landouzy-Dejerine, scapulohumeral, limb-girdle,Von Graefe-Fuchs, oculopharyngeal, myotonic and congenital. In addition,such molecules may be of use in the treatment of congenital (centralcore, nemaline, centronuclear and congenital fiber-type disproportion)and acquired (toxic, inflammatory) myopathies. The present inventionfurther provides for a method of treating a muscle cell disordercomprising administering to the patient an effective amount of NTNRαprotein or an active portion thereof.

[0173] In a further embodiment of the invention, patients that sufferfrom an excess of NTNR, hypersensitivity to NTN, excess NTN, etc. may betreated by administering an effective amount of anti-sense RNA oranti-sense oligodeoxyribonucleotides corresponding to the NTNRα genecoding region thereby decreasing expression of NTNR.

[0174] The compounds and methods of the invention may have use inconditions associated with a decrease in hematopoietic cells. Examplesof these diseases include: anemia (including macrocytic and aplasticanemia); thrombocytopenia; hypoplasia; disseminated intravascularcoagulation (DIC); myelodysplasia; immune (autoimmune) thrombocytopenicpurpura (ITP); and HIV induced ITP. Additionally, these NTNRα moleculesmay be useful in treating myeloproliferative thrombocytotic diseases aswell as thrombocytosis from inflammatory conditions and in irondeficiency. NTNRα polypeptide and NTNRα gene which lead to an increasein hematopoietic cell proliferation may also be used to enhancerepopulation of mature blood cell lineages in cells having undergonechemo- or radiation therapy or bone marrow transplantation therapy.Generally, the NTNRα molecules are expected to lead to an enhancement ofthe proliferation and/or differentiation (but especially proliferation)of hematopoietic cells. Preferred embodiments provide for treatment toenhance hematopoiesis occurring in the spleen.

[0175] Other potential therapeutic applications for NTNRα and NTNRα geneinclude treatment to promote kidney or liver cell growth, survival, andrepair. For example, acute renal failure refers to the abrupt disruptionof previously normal kidney function. This serious clinical condition isdue to a wide variety of mechanisms including circulatory failure(shock), vascular blockage, glomerulonephritis, and obstruction to urineflow. Acute renal failure frequently arises as a complication ofabdominal or vascular surgery. Also, low birth weight, high-riskneonates may now survive lung and heart problems due to continuedimprovements in prenatal care, only to die from complications of acuterenal failure caused by infection or drug toxicity. Of particularclinical importance are cases of acute renal failure associated withtrauma, sepsis, postoperative complications, or medication, particularlyantibiotics. In particular, the compounds of the invention find use inetiologies, directly or indirectly, related to dysfunction of theenteric nervous system or renal system. Specific conditions affectingthe GI include but are not limited to Achalasia, Esophageal spasm,Scleroderma (related to muscular atrophy of the smooth muscle portion ofthe esophagus, weakness of contraction of the lower two-thirds of theesophageal body, and incompetence of the lower esophageal sphincter, butalso caused by treatment with immunosuppressive agents), disorders suchas duodenal ulcer, Zollinger-Ellison Syndrome (hypersecretion of acidcaused by factors including genetic factors, smoking, neuralinfluences), hypersecretion of gastric acid, malabsorptive disorder forexample, in diabetes (and hypoparathyroidism, hyperthyroidism, andadrenal insufficiency) where gastric atony, nausea, vomiting, etc. areat least in part related to dysfunction of thesympatheticlparasympathetic nervous system. Additional disorders includedisorders of intestinal motility, including:Diverticulosis/diverticulitis; Hirschsprung's disease (a congenitaldisorder caused by absence of ganglion cells (Meissner's and Auerbach'splexuses) in a small segment of the distal colon, usually near the anus,typically presented in infants, but in less severe cases, may not bediagnosed until adolescence or early adulthood; Megacolon of other types(Hirschsprung's is a type of megacolon); Intestinal pseudo-obstruction,acute or chronic, which is a severe dysmotility due to abnormalities ofsympathetic innervation of the muscle layers of the intestine, orsecondarily may result from scleroderma, diabetes, amyloidosis, otherneurologic diseases, drugs, or sepsis; chronic constipation, which is aserious problem in patients with mental retardation or neurologicaldiseases, wherein a contributing factor is disordered gut motility. Alsoinclude are treatments for kidney diseases and disorders. Additionalconditions include but not limited to: Spinal cord dysfunction, due toan obvious disruption of enteric nervous system; Guillain Barresyndrome; Multiple sclerosis; Pandysautonomia (dysfunction of autonomicnervous system); Parkinsonism (frequently associated with disorderedgastrointestinal motility); Multiple System Atrophy (Shy DragerSyndrome), which has been documented to have as a feature disordered gutmotility; and porphyria and amyloidosis which are diffuse diseasesmanifested by neuropathy and often with accompanying GI motilitydisorders.

[0176] The necrosis or damage of NTNR-expressing or NTN-responsivetissue includes a necrosis due to microbiologic infection such as vitalhepatitis, tuberculosis, typhoid fever, tularemia, brucellosis, yellowfever, and the like, or necrosis due to ischemic injury resulting fromshock, heart failure, and the like, or necrosis due to acute or chronicreaction with drugs and toxic substances such as chloroform, carbontetrachloride, phosphorous poisoning, and the like. As taught hereincellular growth enhancement, including renal cells such as renalepithelial cells and neuron innervating the kidney, is useful intreating kidney disease. The compounds and methods of the presentinvention provide for the repair of kidney damage. Not to be bound bytheory, it is believed that this can be accomplished, either directly orindirectly, by stimulating kidney cells, including innervating neurons,to grow and divide. Accordingly, a method for regenerating kidney tissueis provided that includes the steps of preparing a NTNRα agonist (e.g.soluble NTNRα optionally complexed with NTN) as disclosed herein,optionally in combination with a pharmacologically acceptable carrier oradditional growth factor or cytokine, and contacting the kidney tissuewith the composition. A therapeutic amount of the composition isadministered. Localized injections or implants are a preferred deliverymethod. Alternatively, damaged kidneys could be removed, treated exvivo, and returned to the host after the kidney is repaired.

[0177] NTNRα agonists, including NTN, can be administered duringhemodialysis. Hemodialysis is defined as the temporary removal of bloodfrom a patient for the purpose of extracting or separating toxinstherefrom and the return of the cleansed blood to the same patient.Hemodialysis is indicated in patients where renal impairment or failureexists, that is, in cases where the blood is not being properly orsufficiently cleansed, (particularly to remove water) by the kidneys. Inthe case of chronic renal impairment or failure, hemodialysis has to becarried out on a repetitive basis. For example, in end stage kidneydisease where transplantation of kidneys is not possible or for medicalreasons is contra-indicated, the patient will have to be dialyzed about100 to 150 times per year. This can result in several thousand accessesto the blood stream to enable the active hemodialysis to be performedover the remaining life of the patient.

[0178] The invention finds use in some immunosuppressive therapies wherethere is the side-effect of kidney damage. For example, therapy of IDDMin humans by methods designed to suppress the autoimmune response.Therapy utilizing cyclosporin A in diabetes can result in kidney damage.The invention finds use in disorders or conditions that can result inkidney damage. For example, diabetes can result in the typical latedamages of blood vessels of the kidneys. Other examples includeimmunologically- or non-immunologically-caused kidney diseases, such ase.g. glomerulonephritis, acute kidney failure, transplant rejection andkidney damage caused by nephrotic substances, kidney transplants, toxicdamage to the kidneys. Furthermore, the present invention finds use inorgan transplantation, including organ transport for storing any organenucleated from a donor to insure the protection of the organ at thetime of its transplantation, minimizing any trouble occurring until thetransplantation operation, and to ensure the preservation of said organin a good condition. A preferred organ is one having NTNR-bearing orNTN-responsive cells. In one specific embodiment the organ is thekidney. Use or intervention with NTNRα agonist, including NTN, promisessuccess with regard to the maintenance of the kidney function.

[0179] As discussed herein, an object of the invention to providemethods for treatment of mammals with dysfunctional gastrointestinalmuscle or disorders of smooth muscles elsewhere in the body. Thegastrointestinal muscle is organized and regulated very differently thanmuscle elsewhere. Both skeletal and smooth muscle in thegastrointestinal tract are under the control of the enteric nervoussystem which is an extremely complex network of nerves and muscles, thatresides within the gastrointestinal wall and orchestrates the entiredigestive process including motility, secretion and absorption. Theenteric nerves are also organized into interconnected networks calledplexuses. Of these, the myenteric plexus, situated between the circularand longitudinal muscle layers, is the main modulator ofgastrointestinal motility. It receives input from both the centralnervous system (via vagal and sympathetic pathways) as well as fromlocal reflex pathways. Its output consists of both inhibitory andexcitatory signals to the adjacent muscle. The final neural pathwayregulating muscle activity in the gastrointestinal tract is thereforerepresented by the neurons of the myenteric plexus. A useful, ifsomewhat simplistic concept is to visualize net muscle tone in thegastrointestinal tract as that resulting from the balance between theopposing effects of two neuronal systems in the myenteric plexus: onecausing the muscle to contract (mainly via acetylcholine) and the othercausing it to relax. Both types of neurons, however, are activated byacetylcholine within the myenteric plexus. The role of acetylcholine inthe regulation of gastrointestinal muscle tone is therefore complex.Acetylcholine directly released by effector nerves near the musclecauses contraction; however, within the plexus, it may result ininhibition or excitation. This is in contrast to skeletal muscle outsidethe gastrointestinal tract which is directly innervated by nervesemanating from the central nervous system. The interaction between nerveand muscle in skeletal muscle outside the gastrointestinal tract is farmore simple: nerves release acetylcholine which causes the muscle tocontract. Finally, the myenteric plexus is probably the most importantbut not the only determinant of muscle tone in the gastrointestinaltract. In fact, basal smooth muscle tone may be visualized as resultingfrom the sum of many different factors including intrinsic (myogenic)tone, and circulating hormones, in addition to nerve activity. It shouldbe clear therefore, that the regulation of gastrointestinal tract musclemotility is far more complex than that of skeletal muscle outside thegastrointestinal tract. While there have been isolated reports on theeffects of botulinum toxin on in vitro preparations of gastrointestinalsmooth muscle, the regulation of gastrointestinal muscle is so complexthat the physiological consequences of blocking neurotransmitter release(by using toxin such as botulinum) in humans or in live animals were notpredictable prior to the present invention. The present inventionprovides compositions, methods, and devices for treatment ofgastrointestinal disorders including achalasia, other disorders of thelower esophageal sphincter, sphincter of Oddi dysfunction, irritablebowel syndrome, and others disorders as discussed herein.

[0180] For example, provided is a method to treat Irritable BowelSyndrome (IBS), which is a motor disorder consisting of altered bowelhabits, abdominal pain, and the absence of detectable pathology. IBS isrecognized by its symptoms, which are markedly influenced bypsychological factors and stressful life situations. IBS is one of themost commonly encountered gastrointestinal disorders. Between 20% and50% of patients referred to gastrointestinal clinics suffer from IBS.Symptoms of IBS occur in approximately 14% of otherwise apparentlyhealthy people. It is a syndrome composed of a number of conditions withsimilar manifestations. The major symptoms of IBS (altered bowel habits,abdominal pain and bloating) are manifestations of increased motility inthe gut and hyper-secretion of gastric acid. Activity of the GI tract ismodulated neurally by the central nervous system (CNS) viaparasympathetic and sympathetic innervation and by the peripherallylocated enteric nervous system (ENS) which resides within the GI tractitself.

[0181] In another aspect is provided the administration of NTNRα to amammal having depressed levels of endogenous NTNRα or a defective NTNRαgene, preferably in the situation where such depressed levels lead to apathological disorder, or where there is lack of activation of the NTNRαand Ret. In these embodiments where the full length NTNRα is to beadministered to the patient, it is contemplated that the gene encodingthe receptor may be administered to the patient via gene therapytechnology.

[0182] In gene therapy applications, genes are introduced into cells inorder to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA, 83:41434146 (1986)). The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

[0183] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, exvivo, or in vivo in the cells of the intended host. Techniques suitablefor the transfer of nucleic acid into mammalian cells in vitro includethe use of liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology, 11:205-210 (1993)). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science,256:808-813 (1992).

[0184] The invention also provides antagonists of NTNRα activation(e.g., NTNRα antisense nucleic acid, neutralizing antibodies).Administration of NTNRα antagonist to a mammal having increased orexcessive levels of endogenous NTNRα activation is contemplated,preferably in the situation where such increased levels of NTNRα or Retactivation lead to a pathological disorder.

[0185] In one embodiment, NTNRα antagonist molecules may be used to bindendogenous ligand in the body, thereby causing desensitized NTNRα tobecome responsive to NTN ligand, especially when the levels of NTNligand in the serum exceed normal physiological levels. Also, it may bebeneficial to bind endogenous NTN ligand which is activating undesiredcellular responses (such as proliferation of tumor cells).

[0186] Pharmaceutical compositions of the soluble NTNRα can furtherinclude a NTN or other NTNRα agonist. Such dual compositions may bebeneficial where it is therapeutically useful to prolong half-life ofNTN, provide a slow-release reservoir for NTN, activate endogenous NTNRαor Ret, and/or to supplement the lack of NTNRα in a targetRet-expressing cell, thereby rendering the cell responsive to NTN.

[0187] Therapeutic formulations of NTNRα are prepared for storage bymixing NTNRα having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter-ions such as sodium; and/or non-ionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

[0188] The NTNRα also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

[0189] NTNRα to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.NTNRα ordinarily will be stored in lyophilized form or in solution.

[0190] Therapeutic NTNRα compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0191] The route of NTNRα administration is in accord with knownmethods, e.g., those routes set forth above for specific indications, aswell as the general routes of injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional means, or sustained release systems asnoted below. NTNRα is administered continuously by infusion or by bolusinjection. Generally, where the disorder permits, one should formulateand dose the NTNRα for site-specific delivery. Administration can becontinuous or periodic. Administration can be accomplished by aconstant- or programmable-flow implantable pump or by periodicinjections.

[0192] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and y ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

[0193] While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated proteins remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for proteinstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0194] Sustained-release NTNRα compositions also include liposomallyentrapped NTNRα. Liposomes containing NTNRα are prepared by methodsknown per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:40304034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal NTNRα therapy.

[0195] When applied topically, the NTNRα is suitably combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bephysiologically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

[0196] For obtaining a gel formulation, the NTNRα formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as PEG to form a gel of theproper viscosity to be applied topically. The polysaccharide that may beused includes, for example, cellulose derivatives such as etherifiedcellulose derivatives, including alkyl celluloses, hydroxyalkylcelluloses, and alkylhydroxyalkyl celluloses, for example,methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxypropyl methylcellulose, and hydroxypropyl cellulose; starch andfractionated starch; agar; alginic acid and alginates; gum arabic;pullullan; agarose; carrageenan; dextrans; dextrins; fructans; inulin;mannans; xylans; arabinans; chitosans; glycogens; glucans; and syntheticbiopolymers; as well as gums such as xanthan gum; guar gum; locust beangum; gum arabic; tragacanth gum; and karaya gum; and derivatives andmixtures thereof. The preferred gelling agent herein is one that isinert to biological systems, nontoxic, simple to prepare, and not toorunny or viscous, and will not destabilize the NTNRα held within it.

[0197] Preferably the polysaccharide is an etherified cellulosederivative, more preferably one that is well defined, purified, andlisted in USP, e.g., methylcellulose and the hydroxyalkyl cellulosederivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose,and hydroxypropyl methylcellulose. Most preferred herein ismethylcellulose.

[0198] The polyethylene glycol useful for gelling is typically a mixtureof low and high molecular weight PEGs to obtain the proper viscosity.For example, a mixture of a PEG of molecular weight 400-600 with one ofmolecular weight 1500 would be effective for this purpose when mixed inthe proper ratio to obtain a paste.

[0199] The term “water soluble” as applied to the polysaccharides andPEGs is meant to include colloidal solutions and dispersions. Ingeneral, the solubility of the cellulose derivatives is determined bythe degree of substitution of ether groups, and the stabilizingderivatives useful herein should have a sufficient quantity of suchether groups per anhydroglucose unit in the cellulose chain to renderthe derivatives water soluble. A degree of ether substitution of atleast 0.35 ether groups per anhydroglucose unit is generally sufficient.Additionally, the cellulose derivatives may be in the form of alkalimetal salts, for example, the Li, Na, K, or Cs salts.

[0200] If methylcellulose is employed in the gel, preferably itcomprises about 2-5%, more preferably about 3%, of the gel and the NTNRαis present in an amount of about 300-1000 mg per ml of gel.

[0201] Semipermeable, implantable membrane devices are useful as meansfor delivering drugs in certain circumstances. For example, cells thatsecrete soluble NTNRα or chimeras can be encapsulated, and such devicescan be implanted into a patient. For example, into the brain of patientssuffering from Parkinson's Disease. See, U.S. Pat. No. 4,892,538 ofAebischer et al.; U.S. Pat. No. 5,011,472 of Aebischer et al.; U.S. Pat.No. 5,106,627 of Aebischer et al.; PCT Application WO 91/10425; PCTApplication WO 91/10470; Winn et al., Exper. Neurology, 113:322-329(1991); Aebischer et al., Exper. Neurology, 111:269-275 (1991); andTresco et al., ASAIO, 38:17-23 (1992). Accordingly, also included is amethod for preventing or treating damage to a nerve or damage to otherNTNR-expressing or NTN-responsive cells, e.g. kidney, as taught herein,which comprises implanting cells that secrete NTNRα, its agonists orantagonists as may be required for the particular condition, into thebody of patients in need thereof. Finally, the present inventionincludes a device for preventing or treating nerve damage or damage toother cells as taught herein by implantation into a patient comprising asemipermeable membrane, and a cell that secretes NTNRα (or its agonistsor antagonists as may be required for the particular condition)encapsulated within said membrane and said membrane being permeable toNTNRα (or its agonists or antagonists) and impermeable to factors fromthe patient detrimental to the cells. The patient's own cells,transformed to produce NTN or NTNRα ex vivo, could be implanted directlyinto the patient, optionally without such encapsulation. The methodologyfor the membrane encapsulation of living cells is familiar to those ofordinary skill in the art, and the preparation of the encapsulated cellsand their implantation in patients may be accomplished without underexperimentation.

[0202] The present invention includes, therefore, a method forpreventing or treating nerve damage by implanting cells, into the bodyof a patient in need thereof, cells either selected for their naturalability to generate NTNRα or engineered to secrete NTNRα. Preferably,the secreted NTNRα being soluble, human mature NTNRα when the patient ishuman. The implants are preferably non-immunogenic and/or preventimmunugenic implanted cells from being recognized by the immune system.For CNS delivery, a preferred location for the implant is the cerebralspinal fluid of the spinal cord.

[0203] An effective amount of NTNRα to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer the NTNRα until a dosage isreached that achieves the desired effect. A typical daily dosage forsystemic treatment might range from about 1 μg/kg to up to 10 mg/kg ormore, depending on the factors mentioned above. As an alternativegeneral proposition, the NTNRα is formulated and delivered to the targetsite or tissue at a dosage capable of establishing in the tissue a NTNRαlevel greater than about 0.1 ng/cc up to a maximum dose that isefficacious but not unduly toxic. This intra-tissue concentration shouldbe maintained if possible by continuous infusion, sustained release,topical application, NTNRα-expressing cell implant, or injection atempirically determined frequencies. The progress of this therapy iseasily monitored by conventional assays. When administered in complexwith or concomitantly with NTN, a 100:1 to 1:100 ratio of NTNRα to NTNdimer is useful. Preferably the ratio is 10:1 to 1 :10, more preferably1:1, and even more preferably 2:1, which may reflect the natural bindingratio of NTNRα to NTN. An amount of NTN effective to produce the desiredresult is administered. A typical daily dosage for systemic treatmentmight also range from about 1 μg/kg to up to 10 mg/kg or more, dependingon the factors mentioned above.

[0204] NTNRα nucleic acid is useful for the preparation of NTNRαpolypeptide by recombinant techniques exemplified herein which can thenbe used for production of anti-NTNRα antibodies having various utilitiesdescribed below.

[0205] The NTNRα (polypeptide or nucleic acid) can be used to increaseNTN-responsiveness (and thus increase cell survival and modulateRet-mediated downstream pathways) of cells in vitro. Such cells mustcontain or be modified to contain cell surface Ret. Cultured ex vivo,these cells may simultaneously be exposed to other known neurotrophicfactors or cytokines, such as those described herein.

[0206] In yet another aspect of the invention, the NTNRα may be used foraffinity purification of ligands that bind to the NTNRα, eithernaturally-occurring or synthetic ligands. NTN is a preferred ligand forpurification. Briefly, this technique involves: (a) contacting a sourceof NTN ligand with an immobilized NTNRα under conditions whereby the NTNligand to be purified is selectively adsorbed onto the immobilizedreceptor; (b) washing the immobilized NTNRα and its support to removenon-adsorbed material; and (c) eluting the NTN ligand molecules from theimmobilized NTNRα to which they are adsorbed with an elution buffer. Ina particularly preferred embodiment of affinity purification, NTNRα iscovalently attaching to an inert and porous matrix or resin (e.g.,agarose reacted with cyanogen bromide). Especially preferred is a NTNRαimmunoadhesin immobilized on a protein A column. A solution containingNTN ligand is then passed through the chromatographic material. The NTNligand adsorbs to the column and is subsequently released by changingthe elution conditions (e.g. by changing pH or ionic strength). Novelligands can be detected by monitoring displacement of a known, labelledNTNRα ligand, such as I¹²⁵- or biotinylated-NTN.

[0207] The NTNRα may be used for competitive screening of potentialagonists or antagonists for binding to the NTNRα. Such agonists orantagonists may constitute potential therapeutics for treatingconditions characterized by insufficient or excessive NTNRα activation,respectively.

[0208] The preferred technique for identifying molecules which bind tothe NTNRα utilizes a chimeric receptor (e.g., epitope-tagged NTNRα orNTNRα immunoadhesin) attached to a solid phase, such as the well of anassay plate. The binding of the candidate molecules, which areoptionally labelled (e.g., radiolabeled), to the immobilized receptorcan be measured. Alternatively, competition for binding of a known,labelled NTNRα ligand, such as I¹²⁵-NTN, can be measured. For screeningfor antagonists, the NTNRα can be exposed to a NTN ligand followed bythe putative antagonist, or the NTN ligand and antagonist can be addedto the NTNRα simultaneously, and the ability of the antagonist to blockreceptor activation can be evaluated.

[0209] The present invention also provides for assay systems fordetecting NTN activity, comprising cells which express high levels ofNTNRα, and which are, therefore, extremely sensitive to even very lowconcentrations of NTN or NTN-like molecules. The present inventionprovides for assay systems in which NTN activity or activities similarto NTN activity resulting from exposure to a peptide or non-peptidecompound may be detected by measuring a physiological response to NTN ina cell or cell line responsive to NTN which expresses the NTNRαmolecules of the invention. A physiological response may comprise any ofthe biological effects of NTN, including but not limited to, thosedescribed herein, as well as the transcriptional activation of certainnucleic acid sequences (e.g. promoter/enhancer elements as well asstructural genes), NTN-related processing, translation, orphosphorylation, the induction of secondary processes in response toprocesses directly or indirectly induced by NTN, including Ret-mediatedeffects, and morphological changes, such as neurite sprouting, or theability to support the survival of cells, for example, nodose or dorsalroot ganglion cells, motoneurons, dopaminergic neurons, sensory neurons,Purkinje cells, or hippocampal neurons.

[0210] In one embodiment of the invention, the functional interactionbetween NTN and the NTNRα may be observed by detecting an increase inthe production autophosphorylated Ret protein, or alternatively,phosphorylated ERK-1 or ERK-2 homologs (See Kotzbauer et al., supra).

[0211] The present invention provides for the development of novel assaysystems which can be utilized in the screening of compounds for NTN- orNTN-like activity. Target cells which bind NTN may be produced bytransfection with NTNRα-encoding nucleic acid or may be identified andsegregated by, for example, fluorescent-activated cell sorting,sedimentation of rosettes, or limiting dilution. Once target cell linesare produced or identified, it may be desirable to select for cellswhich are exceptionally sensitive to NTN. Such target cells may bear agreater number of NTNRα molecules; target cells bearing a relativeabundance of NTNRα can be identified by selecting target cells whichbind to high levels of NTN, for example, by marking high-expressors withfluorophore tagged-NTN followed by immunofluorescence detection and cellsorting. Alternatively, cells which are exceptionally sensitive to NTNmay exhibit a relatively strong biological response in response to NTNbinding, such as a sharp increase in Ret-mediated effects or inimmediate early gene products such as c-fos or c-jun. By developingassay systems using target cells which are extremely sensitive to NTN,the present invention provides for methods of screening for NTN orNTN-like activity which are capable of detecting low levels of NTNactivity.

[0212] In particular, using recombinant DNA techniques, the presentinvention provides for NTN target cells which are engineered to behighly sensitive to NTN. For example, the NTN-receptor gene can beinserted into cells which are naturally NTN responsive such that therecombinant NTNRα gene is expressed at high levels and the resultingengineered target cells express a high number of NTNRs on their cellsurface. Alternatively, or additionally, the target cells may beengineered to comprise a recombinant gene which is expressed at highlevels in response to NTN/receptor binding. Such a recombinant gene maypreferably be associated with a readily detectable product. For example,and not by way of limitation, transcriptional control regions (i.e.promoter/enhancer regions) from an immediate early gene may be used tocontrol the expression of a reporter gene in a construct which may beintroduced into target cells. The immediate early gene/reporter geneconstruct, when expressed at high levels in target cells by virtue of astrong promoter/enhancer or high copy number, may be used to produce anamplified response to NTNRα binding. For example, and not by way oflimitation, a NTN-responsive promoter may be used to control theexpression of detectable reporter genes including β-galactosidase,growth hormone, chloramphenicol acetyl transferase, neomycinphosphotransferase, luciferase, or β-glucuronidase. Detection of theproducts of these reporter genes, well known to one skilled in the art,may serve as a sensitive indicator for NTN or NTN-like activity ofpharmaceutical compounds.

[0213] The NTNRα-encoding or reporter gene constructs discussed herein(e.g., soluble ECD) can be inserted into target cells using any methodknown in the art, including but not limited to transfection,electroporation, calcium phosphate/DEAE dextran methods, and cell gun.The constructs and engineered target cells can be used for theproduction of transgenic animals bearing the above-mentioned constructsas transgenes, from which NTNRα-expressing target cells may be selectedusing the methods discussed.

[0214] Nucleic acids which encode NTNR, preferably from non-humanspecies, such as murine or rat protein, can be used to generate eithertransgenic animals or “knock out” animals which, in turn, are useful inthe development and screening of therapeutically useful reagents. Atransgenic animal (e.g., a mouse) is an animal having cells that containa transgene, which transgene was introduced into the animal or anancestor of the animal at a prenatal, e.g., an embryonic, stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops. In one embodiment, the human and /orrat cDNA encoding NTNRα, or an appropriate sequence thereof, can be usedto clone genomic DNA encoding NTNRα in accordance with establishedtechniques and the genomic sequences used to generate transgenic animalsthat contain cells which express DNA encoding NTNR. Methods forgenerating transgenic animals, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for NTNRα transgene incorporation with tissue-specificenhancers, which could result in desired effect of treatment. Transgenicanimals that include a copy of a transgene encoding NTNRα introducedinto the germ line of the animal at an embryonic stage can be used toexamine the effect of increased expression of DNA encoding NTNR. Suchanimals can be used as tester animals for reagents thought to conferprotection from, for example, diseases related to NTN. In accordancewith this facet of the invention, an animal is treated with the reagentand a reduced incidence of the disease, compared to untreated animalsbearing the transgene, would indicate a potential therapeuticintervention for the disease.

[0215] Transgenic mice bearing minigenes are currently preferred. Firsta fusion enzyme expression construct is created and selected based onexpression in cell culture as described in the Examples. Then a minigenecapable of expressing that fusion enzyme is constructed using knowntechniques. Particularly preferred hosts are those bearing minigeneconstructs comprising a transcriptional regulatory element that istissue-specific for expression.

[0216] Transgenic mice expressing NTNRα minigene are made using knowntechniques, involving, for example, retrieval of fertilized ova,microinjection of the DNA construct into male pronuclei, andre-insertion of the fertilized transgenic ova into the uteri ofhormonally manipulated pseudopregnant foster mothers. Alternatively,chimeras are made using known techniques employing, for example,embryonic stem cells (Rossant et al., Philos. Trans. R. Soc. Lond. BioL339:207-215 (1993)) or primordial germ cells (Vick et al. Philos. Trans.R. Soc. Lond. BioL 251:179-182 (1993)) of the host species. Insertion ofthe transgene can be evaluated by Southern blotting of DNA prepared fromthe tails of offspring mice. Such transgenic mice are then back-crossedto yield homozygotes.

[0217] It is now well-established that transgenes are expressed moreefficiently if they contain introns at the 5′ end, and if these are thenaturally occurring introns (Brinster et al. Proc. Natl. Acad. Sci. USA85:836 (1988); Yokode et al., Science 250:1273 (1990)).

[0218] Transgenic mice expressing NTNRα minigene are created usingestablished procedures for creating transgenic mice. Transgenic mice areconstructed using now standard methods (et al. Proc. Natl. Acad. Sci.USA 85:836 (1988); Yokode et al., Science 250:1273 (1990); Rubin et al.,Proc Natl Acad Sci USA 88:434 (1991); Rubin et al. Nature 353:265(1991)). Fertilized eggs from timed matings are harvested from theoviduct by gentle rinsing with PBS and are microinjected with up to 100nanoliters of a DNA solution, delivering about 10⁴ DNA molecules intothe male pronucleus. Successfully injected eggs are then re-implantedinto pseudopregnant foster mothers by oviduct transfer. Less than 5% ofmicroinjected eggs yield transgenic offspring and only about ⅓ of theseactively express the transgene: this number is presumably influenced bythe site at which the transgene enters the genome.

[0219] Transgenic offspring are identified by demonstratingincorporation of the microinjected transgene into their genomes,preferably by preparing DNA from short sections of tail and analyzing bySouthern blotting for presence of the transgene (“Tail Blots”). Apreferred probe is a segment of a minigene fusion construct that isuniquely present in the transgene and not in the mouse genome.Alternatively, substitution of a natural sequence of codons in thetransgene with a different sequence that still encodes the same peptideyields a unique region identifiable in DNA and RNA analysis. Transgenic“founder” mice identified in this fashion are bred with normal mice toyield heterozygotes, which are back-crossed to create a line oftransgenic mice. Tail blots of each mouse from each generation areexamined until the strain is established and homozygous. Eachsuccessfully created founder mouse and its strain vary from otherstrains in the location and copy number of transgenes inserted into themouse genome, and hence have widely varying levels of transgeneexpression. Selected animals from each established line are sacrificedat 2 months of age and the expression of the transgene is analyzed byNorthern blotting of RNA from liver, muscle, fat, kidney, brain, lung,heart, spleen, gonad, adrenal and intestine.

[0220] Alternatively, the non-human homologs of NTNRα can be used toconstruct a NTNRα “knock out” animal, i.e., having a defective oraltered gene encoding NTNR, as a result of homologous recombinationbetween the endogenous NTNRα gene and an altered genomic NTNRα DNAintroduced into an embryonic cell of the animal. For example, murineNTNRα cDNA can be used to clone genomic NTNRα DNA in accordance withestablished techniques. A portion of the genomic NTNRα DNA (e.g., suchas an exon which encodes e.g., an extracellular domain) can be deletedor replaced with another gene, such as a gene encoding a selectablemarker which can be used to monitor integration. Typically, severalkilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas and Capecchi, Cell 51:503(1987) for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., Li et al.,Cell 69: 915 (1992)). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for their ability to accept grafts, reject tumors anddefend against infectious diseases and can be used in the study of basicimmunobiology.

[0221] In addition to the above procedures, which can be used forpreparing recombinant DNA molecules and transformed host animals inaccordance with the practices of this invention, other known techniquesand modifications thereof can be used in carrying out the practice ofthe invention. For example, U.S. Pat. No. 4,736,866 discloses vectorsand methods for production of a transgenic non-human eukaryotic animalwhose germ cells and somatic cells contain a gene sequence introducedinto the animal, or an ancestor of the animal, at an embryonic stage.U.S. Pat. No. 5,087,571 discloses a method of providing a cell culturecomprising (1) providing a transgenic non-human mammal, all of whosegerm cells and somatic cells contain a recombinant gene sequenceintroduced at an embryonic stage; and (2) culturing one or more of saidsomatic cells. U.S. Pat. No. 5,175,385 discloses vectors and methods forproduction of a transgenic mouse whose somatic and germ cells containand express a gene at sufficient levels to provide the desired phenotypein the mouse, the gene having been introduced into said mouse or anancestor of said mouse at an embryonic stage, preferably bymicroinjection. A partially constitutive promoter, the metallothioneinpromoter, was used to drive heterologous gene expression. U.S. Pat. No.5,175,384 discloses a method of introducing a transgene into an embryoby infecting the embryo with a retrovirus containing the transgene. U.S.Pat. No. 5,175,383 discloses DNA constructs having a gene, homologous tothe host cell, operably linked to a heterologous and inducible promotereffective for the expression of the gene in the urogenital tissues of amouse, the transgene being introduced into the mouse at an embryonicstage to produce a transgenic mouse. Even though a homologous gene isintroduced, the gene can integrate into a chromosome of the mouse at asite different from the location of the endogenous coding sequence. Thevital MMTV promoter was disclosed as a suitable inducible promoter. U.S.Pat. No. 5,162,215 discloses methods and vectors for transfer of genesin avian species, including livestock species such as chickens, turkeys,quails or ducks, utilizing pluripotent stem cells of embryos to producetransgenic animals. U.S. Pat. No. 5,082,779 discloses pituitary-specificexpression promoters for use in producing transgenic animals capable oftissue-specific expression of a gene. U.S. Pat. No. 5,075,229 disclosesvectors and methods to produce transgenic, chimeric animals whosehemopoietic liver cells contain and express a functional gene driven bya liver-specific promoter, by injecting into the peritoneal cavity of ahost fetus the disclosed vectors such that the vector integrates intothe genome of fetal hemopoietic liver cells.

[0222] Although some of the above-mentioned patents and publications aredirected to the production or use of a particular gene product ormaterial that are not within the scope of the present invention, theprocedures described therein can easily be modified to the practice ofthe invention described in this specification by those skilled in theart of fermentation and genetic engineering.

[0223] Assay systems of the present invention enable the efficientscreening of pharmaceutical compounds for use in the treatment ofNTN-associated diseases. For example, and not by way of limitation, itmay be desirable to screen a pharmaceutical agent for NTN activity andtherapeutic efficacy in cerebellar degeneration. In a one embodiment ofthe invention, cells responsive to NTN may be identified and isolated,and then cultured in microwells in a multiwell culture plate. Culturemedium with added test agent, or added NTN, in numerous dilutions may beadded to the wells, together with suitable controls. The cells may thenbe examined for improved survival, neurite sprouting, and the like, andthe activity of test agent and NTN, as well as their relativeactivities, can be determined. For example, one can now identifyNTN-like compounds which can, like NTN, prevent motoneuron cell death inresponse to toxic assault or axotomy, for example. NTN responsivemotoneurons could be utilized in assay systems to identify compoundsuseful in treating motoneuron diseases. If a particular disease is foundto be associated with a defective NTN response in a particular tissue, arational treatment for the disease would be supplying the patient withexogenous NTN. However, it may be desirable to develop molecules whichhave a longer half-life than endogenous NTN, or which act as NTNagonists, or which are targeted to a particular tissue. Accordingly, themethods of the invention can be used to produce efficient and sensitivescreening systems which can be used to identify molecules with thedesired properties. Similar assay systems could be used to identify NTNantagonists.

[0224] In addition, the present invention provides for experimentalmodel systems for studying the physiological role of NTN and itsreceptor. Such systems include animal models, such as (i) animalsexposed to circulating NTNRα peptides which compete with cellularreceptor for NTN binding and thereby produce a NTN-depleted condition,(ii) animals immunized with NTNR; (iii) transgenic animals which expresshigh levels of NTNRα and therefore are hypersensitive to NTN; and (iv)animals derived using embryonic stem cell technology in which theendogenous NTNRα genes were deleted from the genome.

[0225] The present invention also provides for experimental modelsystems for studying the physiological role of NTN and its receptor. Inthese model systems NTNRα protein, peptide fragment, or a derivativethereof, may be either supplied to the system or produced within thesystem. Such model systems could be used to study the effects of NTNexcess or NTN depletion. The experimental model systems may be used tostudy the effects of increased or decreased response to NTN in cell ortissue cultures, in whole animals, in particular cells or tissues withinwhole animals or tissue culture systems, or over specified timeintervals (including during embryogenesis) in embodiments in which NTNRαexpression is controlled by an inducible or developmentally regulatedpromoter. In a particular embodiment of the invention, the CMV promotermay be used to control expression of NTNRα in transgenic animals.Transgenic animals, as discussed herein, are produced by any methodknown in the art, including, but not limited to microinjection, cellfusion, transfection, and electroporation.

[0226] The present invention also provides for model systems forautoimmune disease in which an autoimmune response is directed towardNTNRα. Such models comprise animals which have been immunized withimmunogenic amounts of NTNRα and preferably found to produce anti-NTNRαantibodies and/or cell-mediated immunity. To produce such a modelsystem, it may be desirable to administer the NTNRα in conjunction withan immune adjuvant.

[0227] For example, and not by way of limitation, an experimental modelsystem may be created which may be used to study the effects of excessNTN activity. In such a system, the response to NTN may be increased byengineering an increased number of NTNRs on cells of the model systemrelative to cells which have not been so engineered. These cells shouldalso express Ret or another signalling molecule capable of interactingwith NTNRα and mediating an NTN signal. It may be preferable to providean increased number of NTNRs selectively on cells which normally expressNTNRs. Cells may be engineered to produce increased numbers of NTNRα byinfection with a virus which carries a NTNRα gene of the invention.Alternatively, the NTNRα gene may be provided to the cells bytransfection. If the model system is an animal, a recombinant NTNRα genemay be introduced into the cells of the animal by infection with a viruswhich carries the NTNRα gene or other means as discussed herein. Forexample, a transgenic animal may be created which carries the NTNRα geneas a transgene. In order to ensure expression of NTNR, the NTNRα geneshould be placed under the control of a suitable promoter sequence. Itmay be desirable to put the NTNRα gene under the control of aconstitutive and/or tissue specific promoter. By increasing the numberof cellular NTNRs, the response to endogenous NTN may be increased. Ifthe model system contains little or no NTN, NTN may be added to thesystem. It may also be desirable to add additional NTN to the modelsystem in order to evaluate the effects of excess NTN activity. Overexpressing NTN (or secreted NTN) may be the preferable method forstudying the effects of elevated levels of NTN on cells alreadyexpressing NTNR. More preferably would be to express NTNRα in all cells(general expression) and determine which cells are then endowed withfunctional responsiveness to NTN, thus allowing the potentialidentification of a second receptor component, if one exists.

[0228] An experimental model system may be created which may be used tostudy the effects of diminished NTN activity. This system may permitidentification of processes or neurons which require NTN, and which mayrepresent potential therapeutic targets. In such a system, the responseto NTN may be decreased by providing recombinant NTNRs which are notassociated with a cell surface or which are engineered so as to beineffective in transducing a response to NTN. For example, NTNRαprotein, peptide, or derivative may be supplied to the system such thatthe supplied receptor may compete with endogenous NTNRα for NTN binding,thereby diminishing the response to NTN. The NTNRα may be a cell freereceptor which is either added to the system or produced by the system.For example, a NTNRα protein which lacks the transmembrane domain may beproduced by cells within the system, such as an anchorless NTNRα thatmay be secreted from the producing cell. Alternatively, NTNRα protein,peptide or derivative may be added to an extracellular space within thesystem. In additional embodiments of the invention, a recombinant NTNRαgene may be used to inactivate or “knock out” the endogenous gene byhomologous recombination, and thus create a NTNRα deficient cell,tissue, or animal. For example, and not by way of limitation, arecombinant NTNRα gene may be engineered to contain an insertionalmutation, for example the neo gene, which inactivates NTNR. Such aconstruct, under the control of a suitable promoter, may be introducedinto a cell, such as an embryonic stem cell, by a technique such astransfection, transduction, injection, etc. Cells containing theconstruct may then be selected by G418 resistance. Cells which lack anintact NTNRα gene may then be identified, e.g. by Southern blotting orNorthern blotting or assay of expression. Cells lacking an intact NTNRαgene may then be fused to early embryo cells to generate transgenicanimals deficient in NTNR. A comparison of such an animal with an animalnot expressing endogenous NTN would reveal that either the twophenotypes match completely or that they do not, implying the presenceof additional NTN-like factors or receptors. Such an animal may be usedto define specific cell populations, e.g., neuronal populations, or anyother in vivo processes, normally dependent upon NTN or its receptor.Thus, these populations or processes may be expected to be effected ifthe animal did not express NTNRα and therefore could not respond to NTN.Alternatively, a recombinant NTNRα protein, peptide, or derivative whichcompetes with endogenous receptor for NTN may be expressed on thesurface of cells within the system, but may be engineered so as to failto transduce a response to NTN binding. The recombinant NTNRα proteins,peptides or derivatives described above may bind to NTN with an affinitythat is similar to or different from the affinity of endogenous NTNRα toNTN. To more effectively diminish the response to NTN, the NTNRαprotein, peptide, or derivative may desirably bind to NTN with a greateraffinity than that exhibited by the native receptor. If the NTNRαprotein, peptide, or derivative is produced within the model system,nucleic acid encoding the NTNRα protein, peptide, or derivative may besupplied to the system by infection, transduction, transfection, etc. oras a transgene. As discussed supra, the NTNRα gene may be placed underthe control of a suitable promoter, which may be, for example, atissue-specific promoter or an inducible promoter or developmentallyregulated promoter. In a specific embodiment of the invention theendogenous NTNRα gene of a cell may be replaced by a mutant NTNRα geneby homologous recombination. In a further embodiment of the invention,NTNRα expression may be reduced by providing NTNRα expressing cells withan amount of NTNRα antisense RNA or DNA effective to reduce expressionof NTNRα protein.

[0229] The polypeptides of the invention also find use as feed additivesfor animals. The nucleic acids of the invention find use in preparingthese polypeptides.

[0230] The NTNRα polypeptides are also useful as molecular weightmarkers. To use a NTNRα polypeptide as a molecular weight marker, gelfiltration chromatography or SDS-PAGE, for example, will be used toseparate protein(s) for which it is desired to determine their molecularweight(s) in substantially the normal way. NTNRα, preferably a solubleNTNR, and other molecular weight markers will be used as standards toprovide a range of molecular weights. For example, phosphorylase b(mw=97,400), bovine serum albumin (mw=68,000), ovalbumin (mw=46,000),trypsin inhibitor (mw=20,100), and lysozyme (mw=14,400) can be used asMW markers. The other molecular weight markers mentioned here can bepurchased commercially from Amersham Corporation, Arlington Heights,Ill. The molecular weight markers are generally labeled to facilitatedetection thereof. For example, the markers may be biotinylated and,following separation, can be incubated with streptavidin-horseradishperoxidase so that the various markers can be detected by lightdetection.

[0231] The purified NTNRα, and the nucleic acid encoding it, may also besold as reagents for mechanism studies of NTNRα and its ligands, tostudy the role of the NTNRα and NTN ligand in normal growth anddevelopment, as well as abnormal growth and development, e.g., inmalignancies. NTNRα probes can be used to identify cells and tissueswhich are responsive to NTN in normal or diseased states. For example, apatient suffering from a NTN-related disorder may exhibit an aberrancyof NTNRα expression. The present invention provides for methods foridentifying cells which are responsive to NTN comprising detecting NTNRαexpression in such cells. NTNRα expression may be evidenced bytranscription of NTNRα mRNA or production of NTNRα protein. NTNRαexpression may be detected using probes which identify NTNRα nucleicacid or protein. One variety of probe which may be used to detect NTNRαexpression is a nucleic acid probe, which may be used to detectNTNR-encoding RNA by any method known in the art, including, but notlimited to, in situ hybridization, Northern blot analysis, or PCRrelated techniques. Another variety of probe which may be used is taggedNTN as discussed herein.

[0232] According to the invention, tagged NTN may be incubated withcells under conditions which would promote the binding or attachment ofNTN to said cells. In most cases, this may be achieved under standardculture conditions. For example, in one embodiment of the invention,cells may be incubated for about 30 minutes in the presence of taggedNTN. If the tag is an antibody molecule, it may be preferable to allowNTN to bind to cells first and subsequently wash cells to remove unboundligand and then add anti-NTN antibody tag. In another embodiment of theinvention, tagged NTN on the surface of NTN-responsive cells, hereaftercalled target cells, may be detected by rosetting assays in whichindicator cells that are capable of binding to the tag are incubatedwith cells bearing tagged-NTN such that they adhere to tagged-NTN on thetarget cells and the bound indicator cells form rosette-like clustersaround NTN-tag bearing cells. These rosettes may be visualized bystandard microscopic techniques on plated cells, or, alternatively, mayallow separation of rosetted and non-rosetted cells by densitycentrifugation. In a preferred specific embodiment of the invention,target cells, such as neuronal cells. In alternative embodiments of theinvention, tagged-NTN on the surface of target cells may be detectedusing immunofluorescent techniques in which a molecule which reacts withthe tag, preferably an antibody, directly or indirectly producesfluorescent light. The fluorescence may either be observed under amicroscope or used to segregate tagged-NTN-bearing cells by fluorescenceactivated cell sorting techniques. The present invention also providesfor methods for detecting other forms of tags, such as chromogenic tagsand catalytic tags. An anti-NTNRα antibody can also be used as a probe.The detection methods for any particular tag will depend on theconditions necessary for producing a signal from the tag, but should bereadily discernible by one skilled in the art.

[0233] NTNRα variants are useful as standards or controls in assays forthe NTNRα for example ELISA, RIA, or RRA, provided that they arerecognized by the analytical system employed, e.g., an anti-NTNRαantibody.

[0234] Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. In that the preferred epitope is in the ECD ofthe NTNRA, it is desirable to use NTNRα ECD or a molecule comprising theECD (e.g., NTNRα immunoadhesin) as the antigen for generation ofpolyclonal and monoclonal antibodies. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

[0235] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining 1 mg or 1 μg of the peptide orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to {fraction(1/10)} the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

[0236] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Thus, themodifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete antibodies.

[0237] For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (Cabilly et al.,supra).

[0238] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

[0239] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0240] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. U.S.A., and SP-2 cells available from the American Type CultureCollection, Rockville, Md. U.S.A. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0241] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0242] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

[0243] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

[0244] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0245] The ability of the MAbs to block binding of NTN to its receptorcan be evaluated by ELISA and bioassay utilizing available reagents(rhNTNr-IgG; a stable transfected CHO cell line expressing NTNRα).Neutralizing activities can also be evaluated by neuronal survivalassay(s).

[0246] NTNR-specific MAbs can be developed as discussed above using forexample, the receptor immunoadhesin and transfected cell line) toinitiate new immunization protocols to generate NTNR-specific MAbs foruse as potential agonists or antagonists, as well as forimmunohistochemistry, immunocytochemistry, and assay development. TheMAbs generated from fusion of the immunized animals can be screened foragonist and antagonist activities by bioassay (e.g., neuron survivalassays, signal transduction/phosphorylation, kidney cell survivalassays) as well as by ELISA and FACS (functional blocking of NTN-NTNRαbinding). Suitable techniques are provided in, for example, Lucas etal., J. Immunol. 145:1415-1422 (1990); Hoogenraad et al. J. Immunol.Methods 6:317-320 (1983); Moks et al., Eur. J. Biochem. 85:1205-1210(1986); Laemmli, Nature (London) 227:680-685 (1970); and, Towbin et al.,Proc Natl Acad Sci USA 76:43504354 (1979).

[0247] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

[0248] In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Mark et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

[0249] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (Cabilly et al., supra;Morrison, et al., Proc. Nat. Acad. Sci. USA, 81:6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

[0250] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

[0251] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0252] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0253] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol.,196:901(1987)). Another method uses a particular framework derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

[0254] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

[0255] Alternatively, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be producedin phage- display libraries (Hoogenboom et al., J. Mol. Biol.,227:381(1991); Marks et al., J. Mol. Biol., 222:581(1991)).

[0256] Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different antigens. BsAbs can be used astumor targeting or imaging agents and can be used to target enzymes ortoxins to a cell possessing the NTNRα. Such antibodies can be derivedfrom full length antibodies or antibody fragments (e.g.F(ab′)₂bispecific antibodies). In accordance with the present invention,the BsAb may possess one arm which binds the NTNRα and another arm whichbinds to a cytokine or another cytokine receptor (or a subunit thereof)such as the receptors for TPO, EPO, G-CSF, IL-4, IL-7, GH, PRL; the α orβ subunits of the IL-3, GM-CSF, IL-5, IL-6, LIF, OSM and CNTF receptors;or the α, β, or γ subunits of the IL-2 receptor complex. For example,the BsAb may bind both NTNRα and gp130.

[0257] Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, published May 13, 1993, and inTraunecker et al., EMBO J., 10:3655-3659 (1991).

[0258] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2, andCH3 regions. It is preferred to have the first heavy-chain constantregion (CH1) containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

[0259] In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690published Mar. 3, 1994. For further details of generating bispecificantibodies see, for example, Suresh et al., Methods in Enzymology,121:210 (1986).

[0260] Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0261] Techniques for generating bispecific antibodies from antibodyfragments have also been described in the literature. The followingtechniques can also be used for the production of bivalent antibodyfragments which are not necessarily bispecific. According to thesetechniques, Fab′-SH fragments can be recovered from E. coli, which canbe chemically coupled to form bivalent antibodies. Shalaby et al., J.Exp. Med., 175:217-225 (1992) describe the production of a fullyhumanized BsAb F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the BsAb. The BsAb thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. See also Rodriguez et al., Int. J. Cancers,(Suppl.) 7:45-50 (1992).

[0262] Various techniques for making and isolating bivalent antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bivalent heterodimers have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. The “diabody”technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993) has provided an alternative mechanism for makingBsAb fragments. The fragments comprise a heavy-chain variable domain(VH) connected to a light-chain variable domain (V_(L)) by a linkerwhich is too short to allow pairing between the two domains on the samechain. Accordingly, the V_(H) and V_(L) domains of one fragment areforced to pair with the complementary V_(L) and V_(H) domains of anotherfragment, thereby forming two antigen-binding sites. Another strategyfor making BsAb fragments by the use of single-chain Fv (sFv) dimers hasalso been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0263] The NTNRα agonists (including NTN) and agonist NTNRα antibodiesof the present invention can be used to enhance splenic hematopoiesis,allowing some repopulation of blood cell lineages in patients havingundergone chemo- or radiation therapy and transplantation. Generally,the antibodies will act to enhance proliferation and/or differentiation(but especially proliferation) of hematopoietic cells in the spleen.Without being bound by theory, NTNRα agonists may act directly as agrowth, survival or differentiation factor for hematopoietic cells inthe spleen and/or may indirectly act on the splenic stromal environment(possibly neurons involved in the splenic innervation) to produceanother factor that is responsible for the maintenance of hematopoieticlineages. In any event, as taught herein NTNRα agonist, including NTN,have therapeutic benefit in facilitating the splenic engraftment of bonemarrow transplants following irradiation or chemotherapy or forstimulating extramedullary hematopoiesis in the spleen (which is normalin rodents, but not normally seen in man) in those conditions wherethere is an increased demand for blood cell production due to anemia(red blood cells), chronic infection (neutrophils), bone marrow failure(all lineages), and immune deficiency (lymphocytes). The agonists maysimilarly be useful for treating diseases characterized by a decrease inblood cells. Examples of these diseases include: anemia (includingmacrocytic and aplastic anemia); thrombocytopenia; hypoplasia; immune(autoimmune) thrombocytopenic purpura (ITP); and HIV induced ITP. Also,the agonists may be used to treat a patient having suffered ahemorrhage.

[0264] Therapeutic applications for NTNRα neutralizing antibodiesinclude the treatment of metabolic disorders and cell tumors at sites ofNTNRα expression, especially those tumors characterized byoverexpression of NTNRα.

[0265] For therapeutic applications, the NTNRα antibodies of theinvention are administered to a mammal, preferably a human, in aphysiologically acceptable dosage form, including those that may beadministered to a human intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The NTNRα antibodiesalso are suitably administered by intratumoral, peritumoral,intralesional, or perilesional routes or to the lymph, to exert local aswell as systemic therapeutic effects.

[0266] Such dosage forms encompass physiologically acceptable carriersthat are inherently non-toxic and non-therapeutic. Examples of suchcarriers include ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms of NTNRαantibodies include polysaccharides such as sodium carboxymethylcelluloseor methylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, PEG, and wood waxalcohols. For all administrations, conventional depot forms are suitablyused. Such forms include, for example, microcapsules, nanocapsules,liposomes, plasters, inhalation forms, nose sprays, sublingual tablets,and sustained-release preparations. The NTNRα antibody will typically beformulated in such vehicles at a concentration of about 0.1 mg/ml to 100mg/ml.

[0267] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theNTNRα antibody, which matrices are in the form of shaped articles, e.g.films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., supra and Langer, supra, orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate (Sidman et al., supra),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated NTNRα antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0268] Sustained-release NTNRα antibody compositions also includeliposomally entrapped antibodies. Liposomes containing the NTNRαantibodies are prepared by methods known in the art, such as describedin Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang etal., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Ordinarily; the liposomes are the small (about200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol.% cholesterol, the selected proportion beingadjusted for the optimal NTNRα antibody therapy. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556.

[0269] For the prevention or treatment of disease, the appropriatedosage of NTNRα antibody will depend on the type of disease to betreated, as defined above, the severity and course of the disease,whether the antibodies are administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the NTNRα antibody, and the discretion of the attending physician.The NTNRα antibody is suitably administered to the patient at one timeor over a series of treatments.

[0270] Depending on the type and severity of the disease, about 1 82g/kg to 15 mg/kg of NTNRα antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

[0271] Animal model are available to assess effects of the compounds andmethod of the invention. For example, to assess the effects of treatingdamaged kidneys with compositions that affect growth (Toback, 1977;Toback et al. 1977), an intravenous injection of 1.0 to 1.1 mg ofmercury per kg of body weight as HgCl2 is given to rats to induce areversible syndrome of acute nonoliguric acute renal failure. After oneday, there are marked increases in serum urea nitrogen concentration(SUN), urinary excretion of sodium and protein, and necrosis of proximaltubular cells. By day two, increases in phospholipid, DNA and RNAsynthesis, and mitotic index indicate that cellular regeneration isunderway. By day three, the SUN reaches a maximum, and squamoidepithelial cells appear on the tubular basement membrane. At day five,the SUN returns to normal, the maximal rate of phospholipid synthesis isreached, and the tubules are repopulated with more mature cells. Theeffects of infusion of a composition of autocrine growth factors onrenal structure is compared with untreated rats and animals infused withvehicle alone during the course of the mercuric chloride-induced acutetubular necrosis syndrome discussed above.

[0272] The NTNRα antibodies of the invention are also useful as affinitypurification agents. In this process, the antibodies against NTNRα areimmobilized on a suitable support, such a Sephadex resin or filterpaper, using methods well known in the art. The immobilized antibodythen is contacted with a sample containing the NTNRα to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the NTNRα,which is bound to the immobilized antibody. Finally, the support iswashed with another suitable solvent, such as glycine buffer, pH 5.0,that will release the NTNRα from the antibody.

[0273] NTNRα antibodies may also be useful in diagnostic assays forNTNRα, e.g., detecting its expression in specific cells, tissues, orserum. For diagnostic applications, antibodies typically will be labeledwith a detectable moiety. The detectable moiety can be any one which iscapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C,³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin;radioactive isotopic labels, such as, e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H; or anenzyme, such as alkaline phosphatase, beta-galactosidase, or horseradishperoxidase.

[0274] Any method known in the art for separately conjugating thepolypeptide variant to the detectable moiety may be employed, includingthose methods described by Hunter et al., Nature, 144:945 (1962); Davidet al., Biochemistry, 13:1014 (1974); Pain et al., J. ImmunoL Meth.,40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).

[0275] The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,Inc., 1987).

[0276] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of NTNRα in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0277] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0278] The following Examples of specific embodiments for carrying outthe present invention are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

[0279] The disclosures of all publications, patents and patentapplications cited herein are hereby incorporated by reference in theirentirety.

EXAMPLES

[0280] Example 1

[0281] Cloning of Human NTNRA

[0282] Eight partial human cDNAs (Genbank accession numbers: R02249 (SEQID NO.: 7), H12981 (SEQ ID NO.: 8), W73681 (SEQ ID NO.: 9), W73633 (SEQID NO.: 10), H05619 (SEQ ID NO.: 11), R02135 (SEQ ID NO.: 12), T03342(SEQ ID NO.: 13), and HSClKA 11 (SEQ ID NO.: 14)) were identified ashaving similarity to the GDNF receptor α component (Jing et al. Cell85:1113-1124 (1996); Treanor et al. Nature 382:80-83 (1996)), but werenot identical to GDNFR sequences. A DNA sequence, determined by aligningthese expressed-sequence-tag (“EST”) cDNA sequences, was extended using5′ and 3′ Marathon RACE reactions (Clonetech Inc.) on human spleen mRNA,using conditions supplied by the manufacturer, to obtain an initial setof human cDNA clones. Additional cDNA clones were identified byscreening a human fetal brain cDNA library (Stratagene) using standardprotocols. Lambda cDNA libraries were plated using standard protocolsand a coding region probe, obtained by PCR amplification of the NTNRαgene using the 3′ and 5′ RACE information, was hybridized to thelibrary. From an alignment of the cloned human cDNA sequences, a fulllength cDNA sequence was obtained (SEQ ID NO: 1), which was referred toas the human Neurturin receptor a (“hNTNRα”) cDNA sequence. Thissequence contained a open reading frame sequence (SEQ ID NO: 2) thatencoded a single 464 amino acid protein sequence (SEQ ID NO: 3), whichwas designated human Neurturin receptor α (“hNTNRα”).

[0283] Human NTNRα (“hNTNRα”) displays an overall 47% similarity at theamino acid level to both hGDNFRα and rGDNFRα.

[0284] hNTNRα, like hGDNFRα, is an extracellular protein that isattached to the outer cell membrane via a glycosyl-phosphatidyl inositol(“GPI”) modification. It has an amino terminal signal peptide forsecretion, 3 potential glycosylation sites, and a stretch of 17 carboxyterminal hydrophobic amino acids preceded by a group of 3 small aminoacids (Gly, Ser, Asn) defining a cleavage/binding site for GPI linkage(FIG. 4; Micanovic et al., Proc. Natl. Acad. Sci. USA, 87:157-161(1990); Moran et al., J. Biol. Chem., 266:1250-1257 (1991)). Theposition of their 30 cysteine residues are completely conserved betweenNTNRα and GDNFRα (FIG. 5). The extracellular domain (“ECD”) is flankedby the signal peptide and the GPI-attachment site.

[0285] Example 2

[0286] Cloning of Rat NTNRα (“rNTNRα”)

[0287] Rat NTNRα (“rNTNRα”) was cloned by screening a rat brain cDNAlibrary (Clonetech) using standard protocols. A full-length human NTNRαprobe was hybridized to the rat cDN library at moderate stringency(e.g., 30% formamide at 42° C., wash in 0.1×SSC at 55° C.). The cDNA,having (SEQ ID NO: 4) and containing the complete open reading frame(SEQ ID NO: 5), was designated as rNTNRα cDNA. The open reading framesequence encoded a single 464 amino acid protein sequence (SEQ ID NO:6), which was designated rat Neurturin receptor a (“rNTNRα”).

[0288] Rat NTNRα and human NTNRα display an overall 94% similarity atthe amino acid level. At the DNA level, a 79% identity was observed. RatNTNRα is 46% identical to both rat GDNFRα and human GDNFRα at theprotein level. Rat NTNRα is 53% identical to rat GDNFRα at the DNAlevel.

[0289] rNTNRα, like rGDNFRα, hGDNFRα, and hNTNRα, is an extracellularprotein that is attached to the outer cell membrane via aglycosyl-phosphatidyl inositol (“GPI”) modification. It has an aminoterminal signal peptide for secretion, 3 potential glycosylation sites,and a stretch of 17 carboxy terminal hydrophobic amino acids preceded bya group of 3 small amino acids (Gly, Ser, Asn) defining acleavage/binding site for GPI linkage (FIG. 4). Surprisingly, theposition of the 30 cysteine residues, which are conserved between humanand rat GDNFRα, are conserved between human and rat NTNRα (FIG. 5).These cysteine residues are completely conserved between NTNTRα andGDNFRα (FIG. 5). The extracellular domain (“ECD”) is flanked by thesignal peptide and the GPI-attachment site.

[0290] Example 3

[0291] Vectors for Expression of Membrane-Bound and Soluble NTNRα

[0292] For mammalian protein expression the complete open reading frameof human NTNRα was amplified using PCR and cloned into a CMV basedexpression vector pRK5 or pRK7. The plasmid was designated pRK-hNTNRαfor the human NTNR.

[0293] To make soluble forms rat and human NTNRα, both rat and humanNTNRα-IgG expression construct were made by cloning the first 432 aminoacids of each receptor (which lacks a GPI linkage site) in front of thehuman Fc sequence. For example, the NTNRα-IgG expression construct wasmade by cloning the first 432 amino acids of the receptor (which lacks aGPI linkage site) in front of the human Fc (IfF2a) sequence. Plasmidswere designated pRK-hNTNRα-IgG for hNTNRα fusion and pRK-rNTNRα-IgG forrat NTNRα fusion. The human gene fusion coding nucleic acid sequence isSEQ ID NO: 15, which encodes the human fusion protein SEQ ID NO: 16.Suitable locations for attachment of structure to the ECD are within, orpreferably C-terminal to, the CFTELTTNIIPG sequence of NTNRα ECD. Therat gene fusion coding nucleic acid sequence is SEQ ID NO: 17, whichencodes the rat fusion protein SEQ ID NO: 18.

[0294] Example 4

[0295] Tissue distribution of NTNRα.

[0296] The tissue distribution of the NTNRα mRNA was examined using insitu hybridization analysis. Its distribution was compared to that ofGDNFRα.

[0297] For in situ hybridization, E15.5 rat embryos were immersion-fixedovernight at 4° C. in 4% paraformaldehyde, then cryoprotected overnightin 15% sucrose. Adult rat brains and spinal cords were frozen fresh. Alltissues were sectioned at 16 um, and processed for in situ hybridizationusing ³³P-UTP labelled RNA probes as described (Davis et al. Science259:1736-1739 (1993)). Sense and antisense probes were derived from theN-terminal region of GDNFRα using T7 polymerase. NTNRα RNA was detectedwith a probe, derived from hNTNRα, designated hNTNRα.T7insitu probe (SEQID NO: 19).

[0298] In the embryonic and adult rat nervous system, mRNA for NTNRα wasfound in the ventral midbrain, where dopaminergic neurons are located,in parts of the ventral spinal cord where motoneurons are located, andin the dorsal root ganglia (DRG) were sensory neurons reside. Inaddition, high levels of NTNRα transcripts were found in tissues such asthe embryonic gut, bladder, cardiac conduction system and diaphragm. Inthe adult rat brain NTNRα was found mainly in the substantia nigra,cortex and olfactory bulb as well as in the dorsal horn of the spinalcord. Although NTNRα was occasionally found in tissues that expressGDNFRα, the two transcripts were most often present adjacent to eachother. For example, in the limb, GDNFRα is expressed in muscle cells,whereas NTNRα is found in the brachial plexus nerve which enervates themuscle. Likewise, in the embryonic bladder, NTNRα is expressed in themuscle layer, while GDNFRα is expressed in the underlying epithelia.Finally, in the gut NTNRα is expressed in the mucosal epithelium, whileGDNFRα is expressed only in the adjacent smooth muscles. This pattern ofexpression is consistent with the notion that NTNRα mediates signalsinside, as well as outside, the nervous system, and suggests distinct,complementary biological roles for NTNRα and GDNFRα.

[0299] Example 5

[0300] NTNRα Specifically Binds NTN

[0301] Equilibrium binding experiments were performed to determine thebinding of NTNRα to NTN. Conditioned media from 293 cells transientlytransfected with either the pRK-hNTNRα-IgG or pRK-rNTNRα-IgG constructsprovided soluble receptor. The conditioned media was incubated withapproximately 5 pM ¹²⁵I human Neurturin, mouse Neurturin, or RGDNF alongwith different concentrations of the appropriate cold ligand in PBScontaining 2 mg/ml BSA (Sigma) and 0.05% Brij 96 (Fluka) for 4 hours atroom temperature. Ligand is in excess over the receptor. Receptor/ligandcomplexes were then incubated with protein A Sepharose CL-4B (Pharmacia)for an additional 1 hour at room temperature. After washing with PBScontaining 0.2 mg/ml BSA, specific perceptible counts were measured. TheIGOR program was used to determine Kd. It was found that both human andmouse ¹²⁵1-NTN can bind to recombinant soluble human NTNRα (data notshown) or soluble rNTNRα protein, specifically and reversibly with anapproximate K_(d) of 10 pM (inset to FIG. 6C). In contrast neither humanNTN (data not shown) nor mouse NTN were able to displace ¹²⁵I-RGDNF fromrGDNFRα (FIG. 6B), and no high affinity binding of human or mouse¹²⁵I-NTN to rGDNFRα was detected (FIG. 6A). Accordingly, NTNspecifically binds NTNRα, interacting with high affinity with NTNRα butnot with rGDNFRα. To further confirm that NTNRα is a specific receptorfor NTN, competition binding experiments were performed using125I-rGDNF. Displaceable high affinity binding of ¹²⁵I-rGDNF torecombinant soluble rGDNFRα was readily observed (K_(d) of 3 pM) (FIG.6B inset); however, binding of ¹²⁵I-rGDNF (iodinated either on tyrosineby the Bolton Hunter method or on lysine using lactoperoxidase) toeither human or rat NTNRα was not detected. GDNF interacts at highaffinity with GDNFRα but not with NTNRα. And, NTN specifically bindsNTNRα. While no high affinity interaction between NTN and GDNFRα wasdetected, a low affinity interaction (Kd>1 nM) was observed when higherconcentrations of unlabeled NTN (10 nm) displaced labelled RGDNF fromGDNFRα.

[0302] Cell based equilibrium binding analysis, performed as described(Treanor et al. Nature 382:80-83 (1996)), confirmed the specificity ofGDNFRα and NTNRα for GDNF and Neurturin, respectively. 293 cells, whichwere transiently transfected with either the full length NTNRαexpression construct (or GDNFRα) or an irrelevant (control) construct,provided membrane-bound receptor. The observed specific associationsbetween GDNF and GDNFRα and between NTN and NTNRα using competitionbinding to cells that express unmodified receptors were in agreementwith the soluble receptor data (data not shown).

[0303] The ligand-binding effect of phosphoinositide-specificphospholipase C (“PIPLC”) treatment of the membrane bound receptor wasdetermined. PIPLC is an enzyme that specifically cleaves GPI linkage(Koke et al. Prot. Express. Purification 2:51-58 (1991); Shukla, LifeSci., 10:1323-1335 (1982); Rhee et al., Science, 244:546-550 (1989)).293 cells, which were transiently transfected with either the fulllength NTNRα expression construct or an irrelevant (control) construct,were incubated with ˜20,000 cpm ¹²⁵I human NTN in the presence of theindicated amounts of PIPLC for 90 minutes at room temperature (FIG. 7A).The cells were washed with ice-cold PBS containing 0.2 mg/ml BSA, afterwhich cell associated ¹²⁵I was measured. Consistent with the predictionthat NTNRα is anchored to the cell surface by a GPI linkage, the bindingof ¹²⁵I-NTN to cells expressing NTNRα was significantly reducedfollowing treatment with (FIG. 7A). Accordingly, NTNRα is a specifichigh affinity GPI-linked receptor for NTN.

[0304] Example 6

[0305] NTNRα Mediates Biological Response to NTN

[0306] To confirm that NTNRα mediates the biological response to NTN,the effect of NTN on the survival of cells that express NTNRα wasdetermined. For survival assays E14 rat motoneurons were isolated,plated, and grown in triplicate wells as described. After addition ofthe indicated growth factors, the number of surviving neurons wasdetermined 72 hours later (FIG. 7B). It was observed that NTN canprevent the death of primary motoneurons (FIG. 7B) that express NTNRα(as determined by in situ hybridization). Moreover, in agreement withthe finding that NTN and GDNF utilize distinct receptors, quantitativedifferences in the survival response of motoneurons to the two factorswas detected: GDNF at saturating concentrations promoted the survival of100% of the BDNF-responsive motoneurons; NTN, whose receptor is sparselydistributed in the embryonic ventral horn were motoneurons reside,prevented the death of only 50% of these cells (FIG. 7B). It was alsoobserved that NTN can prevent the death of primary embryonicdopaminergic neurons that express NTNRα.

[0307] To further confirm that NTNRα is a required mediator of the NTNsignal, embryonic motoneurons were treated with PIPLC and their survivalin the presence of NTN or BDNF was monitored in culture. E14 ratmotoneurons were isolated, plated, and grown in triplicate wells asdescribed. PIPLC (2-4 ug/ml) was added to the indicated samples 1-2 hprior to, as well as 12h and 24h following, addition of the indicatedgrowth factors, and the number of surviving neurons was determined 72hlater (FIG. 7C). The number of embryonic rat spinal motoneurons thatremained alive at saturating concentrations of NTN was reduced by 70-90%following PIPLC treatment, whereas no decrease in the response ofPIPLC-treated neurons to brain-derived neurotrophic factor (BDNF) wasnoted. Moreover, when NTN was added to these motoneurons in combinationwith soluble NTNRα (IgG fusion), the response to NTN was restored (FIG.7C). Accordingly, NTNRα appears to be an essential component of the NTNsignaling cascade and has the properties expected of the ligand-bindingsubunit of a functional NTN receptor. In addition, the soluble NTNRαreceptor can impart a NTN-responsiveness to cells that lack cell surfaceNTNRα, but express the complementing Ret transmembrane protein.

[0308] Example 7

[0309] NTNRα Can Act Via Ret

[0310] Since NTNRα, like GDNFRα, lacks a cytoplasmic domain and appearsto be anchored to the outer surface of the cell via GPI, transmission ofthe NTN signals to the cell interior must involve additional proteins.As the tyrosine kinase receptor Ret, which by itself does not bind GDNFor NTN with a high affinity (Jing et al. Cell 85:1113-1124 (1996));Treanor et al. Nature 382:80-83 (1996); data not shown), appears to be asignaling component of the GDNF receptor, Ret transduction of the NTNresponse following binding of NTN to NTNRα was determined. To assay fortyrosine phosphorylation, cells were incubated for 1h at 37° C. with orwithout PIPLC, and then exposed to various concentrations of NTN. Cellswere then removed from the plates with 2 mM EDTA in PBS and lysed withice-cold buffer (10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 1% NP40,5 mM EDTA, 100 mM sodium vanadate, 2 mM PMSF, and 0.2 units ofaprotinin), and used for immunoprecipitation with antisera raisedagainst the 19 amino acid carboxyl terminus of Ret, followed by bindingto protein A sepharose. The immunoprecipitated proteins were released byboiling in SDS sample buffer, separated on an 8% SDS-polyacrylamide gel,transferred to a nitrocellulose membrane, and reacted withanti-phosphotyrosine antibody (Upstate Biotechnology, Inc.); detectionwas with an ECL Western blotting detection system (Amersham LifeScience). The human neuroblastoma cell line, TGW-1, which expressesendogenous c-ret (Ikeda etal. Oncogene 5:1291 (1990); Takahashi et al.Oncogene 6:297 (1991)), was exposed to NTN for 5 minutes, and the levelof Ret tyrosine phosphorylation was determined. NTN clearly inducedphosphorylation of Ret (FIG. 7D), as well as of the receptor tyrosinekinase responsive, cytoplasmic kinase ERK (i.e., MAPK) in this cell line(FIG. 7E), but not in 4 other neuroblastoma lines that were examined(data not shown). Furthermore, consistent with the hypothesis that NTNRαis an essential mediator between NTN and Ret, NTN failed to inducesignificant tyrosine phosphorylation on Ret in cells that were treatedwith PIPLC (FIG. 7F). A similar result was obtained with GDNF.Tyrosine-phosphorylated RET protein was readily detected inPIPLC-treated TGW-1 cell when NTN was added together with a solubleNTNRα.

[0311] Since these findings herein suggested that Ret participates inthe transmission of the NTN signal, it was determined whether Ret ispart of a putative NTN receptor complex. To examine the formation ofprotein complexes upon exposure to NTN, co-immunoprecipitationexperiments were done NTNRα-expressing TGW-1 cells exposed to 500 ng/mlof NTN. After exposure, cells were lysed a mild detergent brij 96(Sigma) (Davis et al. 1993). For mammalian protein expression thecomplete open reading frame was amplified using PCR and cloned into aCMV based expression vector. For co-precipitation experiments, anepitope tag was inserted between the signal peptide and the maturecoding sequence of NTNRα. When protein complexes were immunoprecipitatedwith a polyclonal antibody to Ret and then analyzed on a western blotusing a polyclonal antibody to NTN, NTN was readilyco-immunoprecipitated by Ret antibodies, which is consistent with thenotion that NTN and Ret physically interact on the cell surface. Toconfirm that NTNRα is part of the NTN/Ret protein complex, humanembryonic kidney 293 cells were transiently transfected with expressionvectors, Ret alone or with a combination of expression vectors for c-retand an epitope tagged NTNRα, exposed to NTN, and lysed with a milddetergent brij 96 (Sigma) (Davis et al. 1993). Putative immune complexeswere immunoprecipitated with a polyclonal antibody against Ret,transferred onto a nitrocellulose filter, and analyzed with a polyclonalantibody against the epitope tagged NTNRα. In agreement with the ideathat NTNRα and Ret can be found in a protein complex, NTNRα, in thepresence, but not in the absence of NTN was readilyco-immunoprecipitated by Ret antibodies. These findings demonstratedthat NTN, NTNRα and Ret can form a complex on the cell surface, that Retand NTNRα are components of a functional NTN receptor, an that NTNRα isan intermediary in the interaction between NTN and Ret.

[0312] To ascertain the potential role of NTNRα in the survivalresponses of developing neurons to neurotrophic factors and compare itsfunction with that of GDNFRα, microinjection was used to introduceexpression plasmids encoding NTNRα and GDNFRα into cultured neurons thatnormally do not survive in response to either GDNF or neurturin.Although neurturin promotes the survival of late fetal rat sympatheticneurons (Kotzbauer, 1996), from a survey of several differentpopulations of neurons, it was found herein that sympathetic neurons ofthe superior cervical sympathetic ganglion (SCG) of postnatal day 4 (P4)mice are not supported by either neurturin or GDNF in culture. Becausethese neurons are also relatively easy to microinject and die rapidly indefined medium lacking neurotrophic factors, they are very useful forexamining the involvement of NTNRα in neuronal survival and neurotrophicfactor responsiveness. Ectopic expression of either NTNRα or GDNFRαalone in SCG neurons had a negligible effect on the survival response ofthese neurons to either GDNF or neurturin (less than 5% survive inmedium containing these factors following injection with either NTNRα orGDNFRα expression plasmids). This suggests that neither GDNFRA nor NTNRαalone are capable of mediating survival responses to GDNF and neurturin.This is consistent with the idea that GPI-linked receptors cannotmediate responses to their ligands without the appropriate transmembranesignaling proteins like Ret in the case of GDNFRα (Treanor et al.,1996); Jing et al., 1996) and gp 130 and LIFRβ in the case of CNTFRα(Davis et al., 1993).

[0313] Ret was reported as an essential signaling component of the GDNFreceptor complex (Treanor et al., 1996; Jing et al., 1996; Trupp et al.,1996; Durbec et al., 1996; Vega, 1996). To see if neurons expressingboth of these receptor components would exhibit specific survivalresponses to GDNF and neurturin, neurons were co-injected withexpression plasmids for Ret and NTNRα or GDNFRα. Ectopic expression ofRet alone had only a small effect on the number of SCG neurons survivingin the presence of neurturin or GDNF (between 10 and 15%). However,neurons co-expressing NTNRα plus Ret had a substantially enhancedsurvival response to neurturin that was significantly greater than thatof neurons expressing Ret alone (p=0.003, t-test, n=6). Likewise,neurons co-expressing GDNFRα plus Ret had substantially enhancedsurvival response to GDNF that was also significantly greater than thatof neurons expressing Ret alone (p=0.0002, t-test, n=9). In contrast,neurons co-expressing GDNFRα plus Ret showed no enhanced survivalresponse to neurturin; there were no more Ret/GDNFRα-expressing neuronssurviving with neurturin that Ret-expressing neurons. Likewise, thenumber of Ret/NTNRα-expressing neurons surviving with GDNF was notsignificantly different from the number of Ret-expressing neuronssurviving with this factor (p=0.2, t-test, n=9). These data confirm theresults reported above.

[0314] Example 8

[0315] NTN Is a Neuron Survival Factor In Vivo

[0316] To determine whether NTN can act to promote survival of midbrainDA neurons, cultures of E14 rat ventral midbrain, enriched for DAneurons, were investigated. This culture system revealed that, likeGDNF, NTN can exert potent actions on the survival of tyrosinehydroxylase-expressing cells of the developing midbrain. The potency andefficacy of NTN was similar to that of GDNF, with NTN displaying a trendtowards promoting survival of greater numbers of cells than seen withmaximally-effective doses of GDNF.

[0317] The ability of NTN to promote survival in vitro of embryonic ratmidbrain DA neurons suggests that NTN can be effective in promotingsurvival of DA neurons in the intact adult brain. As indicated herein,the tyrosine kinase Ret is a critical component of both GDNF and NTNreceptor complexes and is expressed in adult rat midbrain DA neurons. Todetermine whether NTNRα is expressed in adult nigral DA neurons, in situhybridization was employed. While sections of the adult rat ventralmidbrain displayed strong signal for GDNFRα, which was largely confinedto the pars compacta of the substantia nigra and the ventral tegmentalarea, a more modest and diffuse signal for signal for NTNRα was observedin the ventral midbrain. In sections stained for tyrosine hydroxylase,the majority of TH+ nigral cells displayed weak or equivocal signal forNTNRα, while intense hybridization was observed for GDNFRα inassociation with TH+ cells of the substantia nigra. Strong signal forNTNRα was, however, observed in regions immediately adjacent to midbrainDA neurons, including cells bordering the dorsolateral aspect of thepars compacta of the substantia nigra, the medial and lateral nuclei ofthe accessory optic tract, and interpeduncular nuclei. Taken with theability of soluble NTNRα expression in and near adult nigral DA neurons,these results suggest that NTN is acting on adult nigral neurons.

[0318] It was determined herein that intranigral injection of NTN canpromote the survival and TH expression of DA neurons following striatal6-OHDA administration, and that the potency and efficacy of NTN issimilar to that of GDNF. In order to independently assess viability andphenotypic expression of nigral DA neurons, cells were counted usingboth a retrograde fluorescent tracer, flurogold, and byimmunocytochemistry for tyrosine hydroxylase. A single intranigralinjection of 1 or 10 ug of NTN administered one week following 6-OHDAadministration lead to nearly 3 fold higher cell viability (as assessedby the retrograde tracer flurogold) at one month post-lesion than seenin vehicle-treated animals. Examination of the number of cellsexpressing tyrosine hydroxylase revealed a significant increase inprotection in rats treated with 10, but not 1 microgram of NTN. Theeffects of single injections of NTN on cell survival and TH expressionwere indistinguishable from that seen with comparable does of GDNF. BothNTN and GDNF can promote the survival of nigral DA neurons followingneurotoxic or traumatic injury.

[0319] As shown herein, NTN is expressed in the developing and adultnigrostriatal system and can exert potent influences on the survival andphenotypic expression of nigral dopaminergic neurons. The actions of NTNto protect DA neurons and promote TH expression following 6-OHDAadministration indicate that this factor can be a useful agent in thetreatment of Parkinson's disease.

[0320] Example 9

[0321] NTNRα and GDNFRA Extracellular Domains Act as Receptor Agonists

[0322] The effects of GDNFRα and NTNRα extracellular domains as receptoragonists were determined by observing survival of ventral mesencephalic,embryonic rat, dopaminergic neurons treated with exogenously addedextracellular domain of either receptor. Cultures enriched fordopaminergic neurons of the ventral mesencephalon were dissected fromE14 rats, in which Day 0 was the day of the first appearance of thevaginal plug. The tissue was treated with enzyme, triturated to a singlecell suspension, and plated as previously described (Poulsen etal.,Neuron 13(5):1245-52 (1994)) with a few exceptions. Cells were plated onglass coverslips, and all growth factors were diluted first into a 20×concentrated solution of 1 mM HCl before the final dilution (resultingin a final concentration of 20 uM HCl in the medium). All factors wereadded once, at the time of plating. The concentration of insulin in themedium was decreased from 5 ug/ml to 2.5 ug/ml. Cultures were plated intriplicate. Either GDNFRα or NTNRα at 1 ug/ml was added to the cultures2 hours after cell plating. At the same time, parallel cultures receivedeither GDNFRα or NTNRα plus 50 ng/ml of either GDNF or NTN, or received50 ng/ml of GDNF or NTN without exogenously added receptor. After 4 daysin culture, the cells were fixed and stained for tyrosine hydroxylase(TH), a marker for dopaminergic neurons, and the number of TH+ cells ineach condition was counted and compared to control cultures (parallelcultures grown in the presence of no added factors). Both the GDNFRα andthe NTNRα extracellular domains contained a histidine tag (6 histidineresidues) at the C-terminus, which provided a convenient handle forpurification of the extracellular domains. The His-tagged-GDNFRα ECDC-terminal is DGLAGASSH HHHHH, where one of the His residues normallypresent in the GDNFR sequence was used to provide part of the tagsequence. The molecules were produced in 293 cells (by transienttransfection, supernatant harvested 96 hr after transfection), purifiedover a Ni-NTA column using standard IMAC purification procedure. Theisolated ECDs were dialyzed into PBS and subsequently stored at 4° C.

[0323] In three separate experiments GDNFRA and NTNRα extracellulardomains, in both the absence and presence of exogenously added ligand,promoted survival of dopaminergic neurons that was significantly greaterthan control cultures (FIGS. 8A to 8C). In each case, the amount ofsurvival was equal or slightly better than the amount of survival withligand (GDNF or NTN) alone. Survival was further increased when bothligand and its particular receptor extracellular domain (e.g. NTN+NTNRa,GDNF+GDNFRα) were added together. Anti-NTN monoclonal antibody added toa control culture (with no added factors) did not result in a decreasein the observed background level of cell growth, indicating thatbackground growth during the test period was not due to endogenous NTN,if present. These results indicate that receptor extracellular domainaddition of this family of receptors, without ligand, promotes asignificant neuron survival effect over control.

[0324] Example 10

[0325] NTNRα and NTN Enhance Dopamine Utilization In Vivo

[0326] It was determined that soluble NTNRα enhances the effects of NTNin the intact adult rat nigrostriatal dopaminergic system.Administration of NTNRα together with NTN increases the ratio of DOPAC(dihydroxyphenylacetic acid, the principal dopamine metabolite in rat)to dopamine, which is indicative of functional upregulation ofdopaminergic neurons. Adult Sprague-Dawley rats (295-345 g, n=23) wereadministered a single 2 μl injection of hNTN (0.1 μg), solublehis-tagged hNTNRα (0.6 μg), both hNTN (0.1 μg) and soluble his-taggedhNTNRα (0.6 μg), or vehicle (4% mannitol, 10 mM HEPES) in the rightstriatum at stereotaxic coordinates 0.5 mm anterior, 3.0 mm lateral tobregma and 4.5 mm ventral to the dura using a 10 μl Hamilton syringewith 26s gauge needle. Seven days after surgery, tissue from selectedbrain regions was harvested for analysis of dopamine and dopaminemetabolite content. Following decapitation of rats, the brains wererapidly removed and immersed in ice cold phosphate-buffered saline for30 seconds, 1 mm coronal sections were cut with the aid of a chilledmetal brain matrix, and tissue punches of three regions of the striatum(anterior, central and posterior), nucleus accumbens, and substantianigra were collected using 11, 13, and 16 gauge needles, respectively.Tissue punches were homogenized in 200 μl 0.1 M perchloric acidcontaining DHBA as an internal standard. Homogenates were centrifuged at23,000-28,000×g for 20 minutes and the supernatants analyzed fordopamine and DOPAC content using ion-pair reverse-phase HPLC withelectrochemical detection. The DOPAC/dopamine ratio was calculated onthe injected and uninjected sides, and expressed as the injected side asa percentage of the uninjected side. As shown in FIG. 9, the combinationof NTN and NTNRα increased DOPAC/dopamine ratio in the striatum comparedto vehicle, NTNRα alone, and NTN alone. These results show that acombination of NTN and NTNRα can potently enhance dopamine utilizationin the striatum, which is useful in the treatment of Parkinson'sdisease.

[0327] Presented herein is a novel sequence encoding a novel receptorfor neurturin, a recently discovered member of the GDNF protein family.The results presented herein reveal the existence of a novel family ofmulti-component receptors for growth and differentiation factors. Thereceptor complex is composed of a shared signaling subunit—thetransmembrane tyrosine kinase receptor Ret—and a GPI-linked,receptor-specific-ligand-binding subunit—NTNRα in the case of NTN, andGDNFRα in the case of GDNF. In view of these findings, further detailson the mechanism of action of NTN and GDNF on the molecular level can bedetermined. In addition, it is now clear that the distinct biologicalactivities of the GDNF/NTN protein family are determined by the distincttissue distribution of their respective ligand-binding-receptorcomponents rather then by the ability to activate different signalingsystems. Finally, the data presented herein provides a contrasting andsimple biological rational for the involvement of what previouslyappeared to be a needlessly complex way to activate a tyrosine kinasereceptor (reviewed in Lindsay and Yancopoulos, Neuron 17:571-574(1996)). It is now apparent that changing the ligand-binding accessorymolecule is a most economic way to recruit the same signalling systemfor usage by multiple growth factors, and that GPI-linked proteins areeconomic ligand-binding accessory molecules. Use of a singletransmembrane signalling system by multiple growth factors appears to beemployed by cytokines (Stahl and Yancopoulos, Ann. Rev. Biophys, Biomol.Struct. 24:269-291 (1993)) as well as by members of the transforminggrowth factor protein family (Wrana et al., Nature 370:341-347 (1994)).Likewise, GPI-linked proteins are used as ligand-binding accessorymolecules in several multi-component receptors, such as the bacterialendotoxin receptor (Lee et al., Proc. Natl. Acad. Sci. USA 90:9930-9934(1993); Pugin et al., Proc. Natl. Acad. Sci. USA 90:2744-2748 (1993))and the receptor for ciliary neurotrophic factor (Davies et al., Science259:1736-1739 (1993)). The discovery of the receptor and associatedreceptor system presented herein defines a novel paradigm in signaltransduction, highlights the diverse strategies which are used totransmit extracellular signals in the vertebrate nervous system, andprovides means for modulating and controlling cell activity and survivalthat expand the treatment modalities available to the clinician.

1 19 2600 base pairs Nucleic Acid Single Linear 1 CCGAGAGCTG CGGGGGGAGGAGGAGGAGGG TGCCGACGCT TGAGTGGGTT 50 CGAGCCCGAG CCGTAGCCGG GGGAGCCAGTCAGTTTCCGG CCAAGGCAGC 100 AGGGAGAAAG ACAAAAAAAC GGTGGGATTT ATTTAACATGATCTTGGCAA 150 ACGTCTTCTT CCTCTTCTTC TTTCTAGACG AGACCCTCCG CTCTTTGGCC200 AGCCCTTCCT CCCTGCAGGA CCCCGAGCTC CACGGCTGGC GCCCCCCAGT 250GGACTGTGTC CGGGCCAATG AGCTGTGTGC CGCCGAATCC AACTGCAGCT 300 CTCGCTACCGCACTCTGCGG CAGTGCCTGG CAGGCCGCGA CCGCAACACC 350 ATGCTGGCCA ACAAGGAGTGCCAGGCGGCC TTGGAGGTCT TGCAGGAGAG 400 CCCGCTGTAC GACTGCCGCT GCAAGCGGGGCATGAAGAAG GAGCTGCAGT 450 GTCTGCAGAT CTACTGGAGC ATCCACCTGG GGCTGACCGAGGGTGAGGAG 500 TTCTACGAAG CCTCCCCCTA TGAGCCGGTG ACCTCCCGCC TCTCGGACAT550 CTTCAGGCTT GCTTCAATCT TCTCAGGGAC AGGGGCAGAC CCGGTGGTCA 600GCGCCAAGAG CAACCATTGC CTGGATGCTG CCAAGGCCTG CAACCTGAAT 650 GACAACTGCAAGAAGCTGCG CTCCTCCTAC ATCTCCATCT GCAACCGCGA 700 GATCTCGCCC ACCGAGCGCTGCAACCGCCG CAAGTGCCAC AAGGCCCTGC 750 GCCAGTTCTT CGACCGGGTG CCCAGCGAGTACACCTACCG CATGCTCTTC 800 TGCTCCTGCC AAGACCAGGC GTGCGCTGAG CGCCGCCGGCAAACCATCCT 850 GCCCAGCTGC TCCTATGAGG ACAAGGAGAA GCCCAACTGC CTGGACCTGC900 GTGGCGTGTG CCGGACTGAC CACCTGTGTC GGTCCCGGCT GGCCGACTTC 950CATGCCAATT GTCGAGCCTC CTACCAGACG GTCACCAGCT GCCCTGCGGA 1000 CAATTACCAGGCGTGTCTGG GCTCTTATGC TGGCATGATT GGGTTTGACA 1050 TGACACCTAA CTATGTGGACTCCAGCCCCA CTGGCATCGT GGTGTCCCCC 1100 TGGTGCAGCT GTCGTGGCAG CGGGAACATGGAGGAGGAGT GTGAGAAGTT 1150 CCTCAGGGAC TTCACCGAGA ACCCATGCCT CCGGAACGCCATCCAGGCCT 1200 TTGGCAACGG CACGGACGTG AACGTGTCCC CAAAAGGCCC CTCGTTCCAG1250 GCCACCCAGG CCCCTCGGGT GGAGAAGACG CCTTCTTTGC CAGATGACCT 1300CAGTGACAGT ACCAGCTTGG GGACCAGTGT CATCACCACC TGCACGTCTG 1350 TCCAGGAGCAGGGGCTGAAG GCCAACAACT CCAAAGAGTT AAGCATGTGC 1400 TTCACAGAGC TCACGACAAATATCATCCCA GGGAGTAACA AGGTGATCAA 1450 ACCTAACTCA GGCCCCAGCA GAGCCAGACCGTCGGCTGCC TTGACCGTGC 1500 TGTCTGTCCT GATGCTGAAA CTGGCCTTGT AGGCTGTGGGAACCGAGTCA 1550 GAATATTTTT GAAAGCTACG CAGACAAGAA CAGCCGCCTG ACGAAATGGA1600 AACACACACA GACACACACA CACCTTGCAA AAAAAAAATT GTTTTTCCCA 1650CCTTGTCGCT GAACCTGTCT CCTCCCAGGT TTCTTCTCTG GAGAAGTTTT 1700 TGTAAACCAAACAGACAAGC AGGCAGGCAG CCTGAGAGCT GGCCCAGGGG 1750 TCCCCTGGCA GGGGAAACTCTGGTGCCGGG GAGGGCACGA GGCTCTAGAA 1800 ATGCCCTTCA CTTTCTCCTG GTGTTTTTCTCTCTGGACCC TTCTGAAGCA 1850 GAGACCGGAC AAGAGCCTGC AGCGGAAGGG ACTCTGGGCTGTGCCTGAGG 1900 CTGGCTGGGG GCAGGACAAC ACAGCTGCTT CCCCAGGCTG CCCACTCTGG1950 GGACCCGCTG GGGGCTGGCA GAGGGCATCG GTCAGCGGGG CAGCGGGGCT 2000GGCCATGAGG GTCCACCTTC AGCCCTTTGG CTTCAAGGAT GGAGATGGTT 2050 TTGCCCTCCCTCTCTGCCCT CGGGTGGGGC TGGTGGGTCT GCAGCTGGTG 2100 TGGGAACTTC CCCACGGATGGCGGTGGAGG GGGTTCGCAC CGTGCTGGGC 2150 TCCCCCTGAC TGTAGCACGG AGTGTTGGGGCTGGGGGCCA GCTCCAGGAG 2200 GGCTTGAGAG CTCAGCCTGC CTGGGAGAGC CCTTGTGGCGAGGCATTAAA 2250 ACTTGGGCAC CAGCTTCTTT CTCGGTGGCA GAAATTTTGA AGTCAGAGAG2300 AAACGGTCCT TTGTTGGCTT CTTTGCTTTC TCGTGGGTCC TTTGGCAGGC 2350CTCCCTTTGG GGAGAGGGAG GGGAGAGACC ACAGCCGGGT GTGTGTCTGC 2400 AGCACCGTGGGCCCTCAAGC TTTCCTGCTG TCTTCTCCCT CCTCCTCCTT 2450 TCCCCTTTCT CTTTCCTCATTTCCTAGACG TACGTCAACT GTATGTACAT 2500 ACCGGGGCTC CTCTCCTAAC ATATATGTATATACACATCC ATATACATAT 2550 ATTGTGTGGT TTCCCCTTTC TTTCCTTTTT TAAGCAACAAAACTATGGGG 2600 1392 base pairs Nucleic Acid Single Linear 2 ATGATCTTGGCAAACGTCTT CTTCCTCTTC TTCTTTCTAG ACGAGACCCT 50 CCGCTCTTTG GCCAGCCCTTCCTCCCTGCA GGACCCCGAG CTCCACGGCT 100 GGCGCCCCCC AGTGGACTGT GTCCGGGCCAATGAGCTGTG TGCCGCCGAA 150 TCCAACTGCA GCTCTCGCTA CCGCACTCTG CGGCAGTGCCTGGCAGGCCG 200 CGACCGCAAC ACCATGCTGG CCAACAAGGA GTGCCAGGCG GCCTTGGAGG250 TCTTGCAGGA GAGCCCGCTG TACGACTGCC GCTGCAAGCG GGGCATGAAG 300AAGGAGCTGC AGTGTCTGCA GATCTACTGG AGCATCCACC TGGGGCTGAC 350 CGAGGGTGAGGAGTTCTACG AAGCCTCCCC CTATGAGCCG GTGACCTCCC 400 GCCTCTCGGA CATCTTCAGGCTTGCTTCAA TCTTCTCAGG GACAGGGGCA 450 GACCCGGTGG TCAGCGCCAA GAGCAACCATTGCCTGGATG CTGCCAAGGC 500 CTGCAACCTG AATGACAACT GCAAGAAGCT GCGCTCCTCCTACATCTCCA 550 TCTGCAACCG CGAGATCTCG CCCACCGAGC GCTGCAACCG CCGCAAGTGC600 CACAAGGCCC TGCGCCAGTT CTTCGACCGG GTGCCCAGCG AGTACACCTA 650CCGCATGCTC TTCTGCTCCT GCCAAGACCA GGCGTGCGCT GAGCGCCGCC 700 GGCAAACCATCCTGCCCAGC TGCTCCTATG AGGACAAGGA GAAGCCCAAC 750 TGCCTGGACC TGCGTGGCGTGTGCCGGACT GACCACCTGT GTCGGTCCCG 800 GCTGGCCGAC TTCCATGCCA ATTGTCGAGCCTCCTACCAG ACGGTCACCA 850 GCTGCCCTGC GGACAATTAC CAGGCGTGTC TGGGCTCTTATGCTGGCATG 900 ATTGGGTTTG ACATGACACC TAACTATGTG GACTCCAGCC CCACTGGCAT950 CGTGGTGTCC CCCTGGTGCA GCTGTCGTGG CAGCGGGAAC ATGGAGGAGG 1000AGTGTGAGAA GTTCCTCAGG GACTTCACCG AGAACCCATG CCTCCGGAAC 1050 GCCATCCAGGCCTTTGGCAA CGGCACGGAC GTGAACGTGT CCCCAAAAGG 1100 CCCCTCGTTC CAGGCCACCCAGGCCCCTCG GGTGGAGAAG ACGCCTTCTT 1150 TGCCAGATGA CCTCAGTGAC AGTACCAGCTTGGGGACCAG TGTCATCACC 1200 ACCTGCACGT CTGTCCAGGA GCAGGGGCTG AAGGCCAACAACTCCAAAGA 1250 GTTAAGCATG TGCTTCACAG AGCTCACGAC AAATATCATC CCAGGGAGTA1300 ACAAGGTGAT CAAACCTAAC TCAGGCCCCA GCAGAGCCAG ACCGTCGGCT 1350GCCTTGACCG TGCTGTCTGT CCTGATGCTG AAACTGGCCT TG 1392 464 amino acidsAmino Acid Linear 3 Met Ile Leu Ala Asn Val Phe Phe Leu Phe Phe Phe LeuAsp Glu 1 5 10 15 Thr Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gln AspPro Glu 20 25 30 Leu His Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala AsnGlu 35 40 45 Leu Cys Ala Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu50 55 60 Arg Gln Cys Leu Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn 6570 75 Lys Glu Cys Gln Ala Ala Leu Glu Val Leu Gln Glu Ser Pro Leu 80 8590 Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu Leu Gln Cys 95 100105 Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu Thr Glu Gly Glu 110 115120 Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser Arg Leu 125 130135 Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr Gly Ala 140 145150 Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 155 160165 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser 170 175180 Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys 185 190195 Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg 200 205210 Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln 215 220225 Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser 230 235240 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg 245 250255 Gly Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp 260 265270 Phe His Ala Asn Cys Arg Ala Ser Tyr Gln Thr Val Thr Ser Cys 275 280285 Pro Ala Asp Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met 290 295300 Ile Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr 305 310315 Gly Ile Val Val Ser Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn 320 325330 Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp Phe Thr Glu Asn 335 340345 Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn Gly Thr Asp 350 355360 Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gln Ala Thr Gln Ala 365 370375 Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp 380 385390 Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Val 395 400405 Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met 410 415420 Cys Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly Ser Asn Lys 425 430435 Val Ile Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Ala 440 445450 Ala Leu Thr Val Leu Ser Val Leu Met Leu Lys Leu Ala Leu 455 460 4643358 base pairs Nucleic Acid Single Linear 4 GCGGTGGCGG CCGCTCTAGAACTAGTGGAT CCCCGGGCTG CAGGAATTCG 50 GCACGAGAGT GAGCCGAGCA AGGGTTAGCGGGAGAAGATT TTTTTTTTTT 100 TGAATCTTTT TCTTCGTCTT GGTGCCAAAG AAGCGACTCTGGTCTCCCGT 150 CCTAGAAGCT CCTACTGGAT TGCTCCTATT CCGTCGGTGG ATTTCTTTCC200 TATTCGCATT TATTCTGACC CCCTCCCTCG CTGCTTCCTT CCAGCCCTTC 250ACTTTCAGAT CGCCTCGCCC CCACCTCTCC AGTCCCCTCC TGGGAAGTGC 300 AGGGGAATTGGACCCACGGG GACTCACGCC TTCCCGGACG CGCGAGCAAA 350 GGGCTGGGCT GACCTCAGGACCAGGCTGTT GGCTTAGAAG GCAGCCAGAC 400 ACATAGCTAC GTGTGTTTGA TTTCAGTGGCAAGGGGGGAC GTCGAGAGGC 450 AGCCCACCGC CCGCCTCCTA CCCCTCCCCC TCCAACCAGCAGTGAGAATC 500 CCAGGACTCG GGATCTTCAA CCGGCGGCCG CCCGGCGGGA TCTCCGCATT550 GGATTTGGGG GTCGTTATTG CTCGGCTGTT ATTATTATCG TTATTTTATT 600TTTATTTTTT AAACCTAAGG GAGAAAGACA CATACACACA AAACTGTGGG 650 ATTTATTTAACATGATCTTG GCAAACGCCT TCTGCCTCTT CTTCTTTTTA 700 GACGAAACCC TCCGCTCTTTGGCCAGCCCT TCCTCCCTGC AGGGCTCTGA 750 GCTCCACGGC TGGCGCCCCC AAGTGGACTGTGTCCGGGCC AATGAGCTGT 800 GTGCGGCTGA ATCCAACTGC AGCTCCAGGT ACCGCACCCTTCGGCAGTGC 850 CTGGCAGGCC GGGATCGCAA TACCATGCTG GCCAATAAGG AGTGCCAGGC900 AGCCCTGGAG GTCTTGCAGG AAAGCCCACT GTATGACTGC CGCTGCAAGC 950GGGGCATGAA GAAGGAGCTG CAGTGTCTGC AGATCTACTG GAGCATCCAT 1000 CTGGGGCTGACAGAGGGTGA GGAGTTCTAT GAAGCTTCCC CCTATGAGCC 1050 TGTGACCTCG CGCCTCTCGGACATCTTCAG GCTCGCTTCA ATCTTCTCAG 1100 GGACAGGGAC AGACCCGGCG GTCAGTACCAAAAGCAACCA CTGCCTGGAT 1150 GCCGCCAAGG CCTGCAACCT GAATGACAAC TGCAAGAAGCTTCGCTCCTC 1200 TTATATCTCC ATCTGCAACC GTGAGATCTC TCCCACCGAA CGCTGCAACC1250 GCCGCAAGTG CCACAAGGCT CTGCGCCAGT TCTTTGACCG TGTGCCCAGC 1300GAGTATACCT ACCGCATGCT CTTCTGCTCC TGTCAGGACC AGGCATGTGC 1350 TGAGCGTCGCCGGCAAACCA TCCTGCCCAG TTGCTCCTAT GAGGACAAGG 1400 AGAAGCCCAA CTGCCTGGACCTGCGCAGCC TGTGTCGTAC AGACCACCTG 1450 TGCCGGTCCC GACTGGCAGA TTTCCACGCCAACTGTCGAG CCTCCTACCG 1500 GACAATCACC AGCTGTCCTG CGGACAACTA CCAGGCATGTCTGGGCTCCT 1550 ATGCTGGCAT GATTGGGTTT GATATGACAC CCAACTATGT GGACTCCAAC1600 CCCACGGGCA TCGTGGTGTC TCCCTGGTGC AATTGTCGTG GCAGTGGGAA 1650CATGGAAGAA GAGTGTGAGA AGTTCCTCAG GGACTTCACG GAAAACCCAT 1700 GCCTCCGGAATGCCATTCAG GCCTTTGGTA ATGGCACAGA TGTGAACATG 1750 TCTCCCAAAG GCCCCTCACTCCCAGCTACC CAGGCCCCTC GGGTGGAGAA 1800 GACTCCTTCA CTGCCAGATG ACCTCAGTGACAGCACCAGC CTGGGGACCA 1850 GTGTCATCAC CACCTGCACA TCTATCCAGG AGCAAGGGCTGAAGGCCAAC 1900 AACTCCAAAG AGTTAAGCAT GTGCTTCACA GAGCTCACGA CAAACATCAG1950 TCCAGGGAGT AAAAAGGTGA TCAAACTTAA CTCAGGCTCC AGCAGAGCCA 2000GACTGTCGGC TGCCTTGACT GCCCTCCCAC TCCTGATGCT GACCTTGGCC 2050 TTGTAGGCCTTTGGAACCCA GCACAAAAGT TCTTCAAGCA ACCCAGATAT 2100 GAACTCCCGC CTGACAAAATGGAAACACAC GCATACACAC ATGCACACAC 2150 ACACAAACAC ACACACACAC ACACACACACACACACACAC ACACACACAC 2200 ACACCCCTTG CAAAAACACT TTTTTTCCTA CATTGTCTCTGAACCTTTCT 2250 CCTCCCAAGT TTCTTCTCTG GAGAAGTTTT TCTAAACCAA ACAGACAAGC2300 AGGCGGGCAG TCAGAAGCCT GCCCAGAGGT CCCCTGCAAG GGACACCCAG 2350CACCAACGAG GGCTCAAGGC TCTTGAGAGA CTCTTTTCTC TTCACTGGTG 2400 TTTTCTCTCTGGACAAGATG AGACCCTGAT GTGGAAGGTA CTTTGCTGTG 2450 CCTGGTGTGG ACTGGGGAAAGGACAGTTGC AGCTGCCTAC TCTGGGGACC 2500 TGCCCAAGGG TTCACAGAGA GTCTCAGTCAGCAAGGAAGC AGGGCTGGCC 2550 ACAAGGACTT TGTCACCTCT TCCTCTTGGC TTCAGAGATGGAAATGGTTT 2600 GCTGCCATCC CCAGCCATTA TGTGGCCTAG TGGGTTTAAG TCTGGAGTAG2650 GAAGCCTCAT GGCAGCTTCA GGCCATGGTG CCTGTAGTAT AGCTGGGGTT 2700GGGAGCTGTT ACAGGAGGAA GCTTCTTTGG GGCATGAGCA AGCCTTGGTT 2750 GGGCACCAGCTCCAAGATGT ACCTTCCTCC TTTATGCCAG GAATCTTGAA 2800 GTCAAAGAGA AATGATCCTCTGTTGGCTCT TTTTTGTTTG TTTTTGAATT 2850 TTTTTGTGGG TCCATTTGGC AGGTCTCTCTTGGGGAGAAG GGCTGTTGAG 2900 TGGGGCTGGG GAGACCCTAG CTGGGCGTGT GTATGGAGCACTCTGGTGGG 2950 TTCCCAAGCT TGCCCCTTCT CTCTTCTTGT TTCTGCTTTC TCTCTCATTT3000 CTGAGACATT CATGCACTGT CTGTACATAC TGGGTCTCCT TTCTCAACAT 3050ATGTGTATAT CCATATCCAT ATATCCTATG ATTTTACTCT TTCTTTCATT 3100 TTTTTTAAAGAAACAAAACT ATGGAAATAA TACCCTACAG ATGAGCGAAA 3150 ATGTATTATT GTAAAGTTTATTTTTTTTAA TAATGTTGTC TATGATGGGA 3200 AGAAAGGTAC CAGGACCCCT GAGCCCTGGTCCAGTTGGGC TGGTGGGGCT 3250 GTGGCCGGGG ACTCCCGATT GCATTCACTC TTAACCAAGCTCCAATAAAC 3300 GTACTAGGAA GCGAAAAAAA AAAAAAAAAA ACTCGAGGGG GGGCCCGGTA3350 CCCAATTC 3358 1392 base pairs Nucleic Acid Single Linear 5ATGATCTTGG CAAACGCCTT CTGCCTCTTC TTCTTTTTAG ACGAAACCCT 50 CCGCTCTTTGGCCAGCCCTT CCTCCCTGCA GGGCTCTGAG CTCCACGGCT 100 GGCGCCCCCA AGTGGACTGTGTCCGGGCCA ATGAGCTGTG TGCGGCTGAA 150 TCCAACTGCA GCTCCAGGTA CCGCACCCTTCGGCAGTGCC TGGCAGGCCG 200 GGATCGCAAT ACCATGCTGG CCAATAAGGA GTGCCAGGCAGCCCTGGAGG 250 TCTTGCAGGA AAGCCCACTG TATGACTGCC GCTGCAAGCG GGGCATGAAG300 AAGGAGCTGC AGTGTCTGCA GATCTACTGG AGCATCCATC TGGGGCTGAC 350AGAGGGTGAG GAGTTCTATG AAGCTTCCCC CTATGAGCCT GTGACCTCGC 400 GCCTCTCGGACATCTTCAGG CTCGCTTCAA TCTTCTCAGG GACAGGGACA 450 GACCCGGCGG TCAGTACCAAAAGCAACCAC TGCCTGGATG CCGCCAAGGC 500 CTGCAACCTG AATGACAACT GCAAGAAGCTTCGCTCCTCT TATATCTCCA 550 TCTGCAACCG TGAGATCTCT CCCACCGAAC GCTGCAACCGCCGCAAGTGC 600 CACAAGGCTC TGCGCCAGTT CTTTGACCGT GTGCCCAGCG AGTATACCTA650 CCGCATGCTC TTCTGCTCCT GTCAGGACCA GGCATGTGCT GAGCGTCGCC 700GGCAAACCAT CCTGCCCAGT TGCTCCTATG AGGACAAGGA GAAGCCCAAC 750 TGCCTGGACCTGCGCAGCCT GTGTCGTACA GACCACCTGT GCCGGTCCCG 800 ACTGGCAGAT TTCCACGCCAACTGTCGAGC CTCCTACCGG ACAATCACCA 850 GCTGTCCTGC GGACAACTAC CAGGCATGTCTGGGCTCCTA TGCTGGCATG 900 ATTGGGTTTG ATATGACACC CAACTATGTG GACTCCAACCCCACGGGCAT 950 CGTGGTGTCT CCCTGGTGCA ATTGTCGTGG CAGTGGGAAC ATGGAAGAAG1000 AGTGTGAGAA GTTCCTCAGG GACTTCACGG AAAACCCATG CCTCCGGAAT 1050GCCATTCAGG CCTTTGGTAA TGGCACAGAT GTGAACATGT CTCCCAAAGG 1100 CCCCTCACTCCCAGCTACCC AGGCCCCTCG GGTGGAGAAG ACTCCTTCAC 1150 TGCCAGATGA CCTCAGTGACAGCACCAGCC TGGGGACCAG TGTCATCACC 1200 ACCTGCACAT CTATCCAGGA GCAAGGGCTGAAGGCCAACA ACTCCAAAGA 1250 GTTAAGCATG TGCTTCACAG AGCTCACGAC AAACATCAGTCCAGGGAGTA 1300 AAAAGGTGAT CAAACTTAAC TCAGGCTCCA GCAGAGCCAG ACTGTCGGCT1350 GCCTTGACTG CCCTCCCACT CCTGATGCTG ACCTTGGCCT TG 1392 464 amino acidsAmino Acid Linear 6 Met Ile Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe LeuAsp Glu 1 5 10 15 Thr Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gln GlySer Glu 20 25 30 Leu His Gly Trp Arg Pro Gln Val Asp Cys Val Arg Ala AsnGlu 35 40 45 Leu Cys Ala Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu50 55 60 Arg Gln Cys Leu Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn 6570 75 Lys Glu Cys Gln Ala Ala Leu Glu Val Leu Gln Glu Ser Pro Leu 80 8590 Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu Leu Gln Cys 95 100105 Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu Thr Glu Gly Glu 110 115120 Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser Arg Leu 125 130135 Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr Gly Thr 140 145150 Asp Pro Ala Val Ser Thr Lys Ser Asn His Cys Leu Asp Ala Ala 155 160165 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser 170 175180 Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys 185 190195 Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg 200 205210 Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln 215 220225 Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser 230 235240 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg 245 250255 Ser Leu Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp 260 265270 Phe His Ala Asn Cys Arg Ala Ser Tyr Arg Thr Ile Thr Ser Cys 275 280285 Pro Ala Asp Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met 290 295300 Ile Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser Asn Pro Thr 305 310315 Gly Ile Val Val Ser Pro Trp Cys Asn Cys Arg Gly Ser Gly Asn 320 325330 Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp Phe Thr Glu Asn 335 340345 Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn Gly Thr Asp 350 355360 Val Asn Met Ser Pro Lys Gly Pro Ser Leu Pro Ala Thr Gln Ala 365 370375 Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp 380 385390 Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Ile 395 400405 Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met 410 415420 Cys Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly Ser Lys Lys 425 430435 Val Ile Lys Leu Asn Ser Gly Ser Ser Arg Ala Arg Leu Ser Ala 440 445450 Ala Leu Thr Ala Leu Pro Leu Leu Met Leu Thr Leu Ala Leu 455 460 464229 base pairs Nucleic Acid Single Linear 7 CCAAGAGCAA CCATTGCCTGGATGCTGCCA AGGCCTGCAA CCTGAATGAC 50 AACTGCAAGA AGCTGCGCTC CTCCTACATCTCCATCTGCA ACCGCGAGAT 100 CTCGCCCACC GAGCGCTGCA ACCGCCGCAA GTGCCACAAGGCCCTGCGCC 150 AGTTCTTCGA CCGGGTGCCC AGCGAGTACA CCTACCGCAT GCTCTTCTGC200 TCCTGCCAAG ATCAGGCGTG CGCTGAGNC 229 521 base pairs Nucleic AcidSingle Linear 8 CAACCATTGC CTGGGATGCT GCCAAGGCCT GCAACCTGAA TGACAACTGC50 AAGAAGCTGC GCTCCTCCTA CATCTCCATC TGCAACCGCG AGATCTCGCC 100 CACCGAGCGCTGCAACCGCC GCAAGTGCCA CAAGGCCCTG CGCCAGTTCT 150 TCGACCGGGT GCCCAGCGAGTACACCTACC GCATGCTCTT CTGCTCCTGC 200 CAAGACCAGG CGTGCGCTGA GCCGCGGNCAAAACCATCCT GCCCAGCTGC 250 TCCTATGAGG ACAAGGAGAA GCCCAACTGC CTGGGACCTGCGTGGCGTGT 300 GCCGGGACTG ACCACCTGTG TCGGTCCCGG CTNGGCCGAC TTTCCATGGC350 CAATTTGTTG GAGCCTTCCT ACCAGACGGG TCANCAGGTT GCCTTGCGGA 400CAATTTACCA GGGGTNTTTT GGGGTTTTTA TTGTTGGGCA TGGATTGGGG 450 TTTTGAAATTGANAATTAAT TTTGTTGGGA TTTNCAGGCC CCATTGGGCN 500 TTGTNGGTGN TTCCCCTGGG G521 478 base pairs Nucleic Acid Single Linear 9 TGACACCTAA CTATGTGGACTCCAGCCCCA CTGGCATCGT GGTGTCCCCC 50 TGGTGCAGCT GTCGTGGCAG CGGGAACATGGAGGAGGAGT GTGAGAAGTT 100 CCTCAGGGAC TTCACCGAGA ACCCATGCCT CCGGAACGCCATCCAGGCCT 150 TTGNAACGGC ACGGACGTGA ACGTGTCCCC AAAAGGCCCC TCGTTCCAGG200 CCACCCAGGC CCTCGGGTGG AGAAGACGCC TTCTTTGCCA GATGACCTCA 250GTGACAGTAC CAGCTTGGGG ACCAGTGTCA TCACCACCTG CACGTCTGTC 300 CAGGAGCAGGGGCTGAAGGC CAACAACTCC AAAGAGTTAA GCATGTGCTT 350 CACAGAGCTC ACCGACAAATATCATCCCAG GGAGTAACAA GGTGATTCAA 400 ACCTAACTCA GGCCCCAGCA GAGCAAGACCGTCGGCTTGC CTTTGACCGT 450 GCTGTCTGTC CTGATGCTGA ACAGGCTT 478 433 basepairs Nucleic Acid Single Linear 10 GCAAGGTGTG TGTGTGTCTG TGTGTGTTTCCATTTCGTCA GGCGGCTGTT 50 CTTGTCTGCG TACTTTCAAA AATCTTCTGA CTCGGTTCCCACAGCCTACA 100 AGGCCTGTTT CAGCATCAGG ACAGACAGCA CGGTCAAGGC AGCCGACGGT150 CTGGCTCTGC TGGGGCCTGA GTTAGGTTTG ATCACCTTGT TACTCCCTGG 200GATGATATTT GTCGTGAGCT CTGTGAAGCA CATGCTTAAC TCTTTGGAGT 250 TGTTGGCCTTCAGCCCCTGC TCCTGGACAG ACGTGCAGGT GGTGATGACA 300 CTGGGTCCCC AAGCTGGTACTGTCACTGAG GTCATCTGGC AAAGAAGGCG 350 TCTTCTCCAC CCGAGGGGCC TGGGGTGGCTGGGAACGAGG GGGCCTTTTT 400 GGGGGACACG TTCACGTTCC GTTGCCGTTG CCA 433 418base pairs Nucleic Acid Single Linear 11 TTTTTTTTTT TGGGAAAAACAATTTTTTTT TTGCAAGGTG TGTGTGTGTC 50 TGTGTGTGTT TCCATTTCGT CAGGCGGCTGTTCTTGTCTG CGTANTTTTC 100 AAAAATCTTC TGACTCGGTT CCCACAGCCT ACAAGGCCTGTTTCAGCATC 150 AGGACAGACA GCACGGTCAA GGCAGCCGAC GGTCTGGCTC TGCTGGGGCC200 TGAGTTAGGT TTGATCACCT TGTTACTCCC TGGGATGATA TTTNTCGTGA 250GCTCTGTGAA GCACATGCTT AACTCTTTGG AGTTNTTGGC CTTCAGCCCC 300 TGCTCCTGGGACAGAACGTG CAGGNTGGGT GATGACACTG GGNCCCCAAG 350 GCTGGGTACT GTCACTGAGGGTCATCTGGN CAAAGNAAGG NCGTTTTTCT 400 CCACCCGAGG GGCCGGGG 418 364 basepairs Nucleic Acid Single Linear 12 TGTGTGTGTC TGTGTGTGTT TCCATTTCGTCAGGCGGCTG TTCTTGTCTG 50 CGTAGTTTCA AAAATCTTCT GACTCGGTTC CCACAGCCTACAAGGNCTGT 100 TTCAGCATCA GGACAGACAG CACGGTCAAG GCAGCCGACG GTCTGGCTCT150 GCTGGGGCCT GAGTTAGGTT TGATCACCTT GTTACTCCCT GGGATGATAT 200TTGTCGTGAG CTCTGTGAAG CACATGCTTA ACTCTTTGGA GTTGTTGGCC 250 TTCAGCCCCTGCTCCTGGAC AGACGTGCAG GTGGTNATGA CACTGGTCCC 300 CAAGCTGGTA CTNTCACTGAGGTCATCTGG CAAAGAAGGC GTCTTCTCCA 350 CCCNAGGGGC CTGG 364 319 base pairsNucleic Acid Single Linear 13 GGGAAAAACA ATTTTATTTT TGCAAGGTGTGTGTGTGTCT GTGTGTGTTT 50 CCATTTCGTC AGGCGGCTGT CCTTGTCTGC GTAGTTTCAAAAATCTTCTG 100 ACTCGGTTCC CACAGCCTAC AAGGCCTGTA TAAGCATCAG GACAGACAGC150 ACGGTCAAGG CAGCCGACGG TCTGGCTCTG CTGGGGCCTG AGTAAGGTTT 200GNCCACCTTG TAACTCCCTG GGATGATATT TGTCGTGAGC NCTGTNANGC 250 ACATGNTTAACTCTTTGGAG TTNTTGGCCT TCAGCCCCTG CCCCTGGNCA 300 GACGTGCAGG TGGTGATGA 319309 base pairs Nucleic Acid Single Linear 14 GCTGAAACTG GCCTTGTAGGCTGTGGGAAC CGAGTCAGAA TATTTTTGAA 50 AGCTACGCAG ACAAGAACNG CGGCCTGACGAAATGGAAAC ACACACAGAC 100 ACACACACNC CTTGCATAAA AAAAATTGTT TTTCCCACCTTGTCGCTGAA 150 CCTGTCTCCT CCCAGGTTTC TTCTCTGGAG AAGTTTTTGT AAACCAAACA200 GACAAGCAGG CAGGCAGCCT GAGAGCTGGC CCAGGGGTCC CCTGGTCAGG 250GGAAACTCTG GTGCCGGGGA GGGCACGTGG CTCTAGAAAT GCCCTTCACT 300 TTCTCCTGG 3091995 base pairs Nucleic Acid Single Linear 15 ATGATCTTGG CAAACGTCTTCTTCCTCTTC TTCTTTCTAG ACGAGACCCT 50 CCGCTCTTTG GCCAGCCCTT CCTCCCTGCAGGACCCCGAG CTCCACGGCT 100 GGCGCCCCCC AGTGGACTGT GTCCGGGCCA ATGAGCTGTGTGCCGCCGAA 150 TCCAACTGCA GCTCTCGCTA CCGCACTCTG CGGCAGTGCC TGGCAGGCCG200 CGACCGCAAC ACCATGCTGG CCAACAAGGA GTGCCAGGCG GCCTTGGAGG 250TCTTGCAGGA GAGCCCGCTG TACGACTGCC GCTGCAAGCG GGGCATGAAG 300 AAGGAGCTGCAGTGTCTGCA GATCTACTGG AGCATCCACC TGGGGCTGAC 350 CGAGGGTGAG GAGTTCTACGAAGCCTCCCC CTATGAGCCG GTGACCTCCC 400 GCCTCTCGGA CATCTTCAGG CTTGCTTCAATCTTCTCAGG GACAGGGGCA 450 GACCCGGTGG TCAGCGCCAA GAGCAACCAT TGCCTGGATGCTGCCAAGGC 500 CTGCAACCTG AATGACAACT GCAAGAAGCT GCGCTCCTCC TACATCTCCA550 TCTGCAACCG CGAGATCTCG CCCACCGAGC GCTGCAACCG CCGCAAGTGC 600CACAAGGCCC TGCGCCAGTT CTTCGACCGG GTGCCCAGCG AGTACACCTA 650 CCGCATGCTCTTCTGCTCCT GCCAAGACCA GGCGTGCGCT GAGCGCCGCC 700 GGCAAACCAT CCTGCCCAGCTGCTCCTATG AGGACAAGGA GAAGCCCAAC 750 TGCCTGGACC TGCGTGGCGT GTGCCGGACTGACCACCTGT GTCGGTCCCG 800 GCTGGCCGAC TTCCATGCCA ATTGTCGAGC CTCCTACCAGACGGTCACCA 850 GCTGCCCTGC GGACAATTAC CAGGCGTGTC TGGGCTCTTA TGCTGGCATG900 ATTGGGTTTG ACATGACACC TAACTATGTG GACTCCAGCC CCACTGGCAT 950CGTGGTGTCC CCCTGGTGCA GCTGTCGTGG CAGCGGGAAC ATGGAGGAGG 1000 AGTGTGAGAAGTTCCTCAGG GACTTCACCG AGAACCCATG CCTCCGGAAC 1050 GCCATCCAGG CCTTTGGCAACGGCACGGAC GTGAACGTGT CCCCAAAAGG 1100 CCCCTCGTTC CAGGCCACCC AGGCCCCTCGGGTGGAGAAG ACGCCTTCTT 1150 TGCCAGATGA CCTCAGTGAC AGTACCAGCT TGGGGACCAGTGTCATCACC 1200 ACCTGCACGT CTGTCCAGGA GCAGGGGCTG AAGGCCAACA ACTCCAAAGA1250 GTTAAGCATG TGCTTCACAG AGCTCACGAC AAATATCATC CCAGGGCCTA 1300GGGACCCGGT GGACAAAACT CACACATGCC CACCGTGCCC AGCACCTGAA 1350 CTCCTGGGGGGACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC 1400 CCTCATGATC TCCCGGACCCCTGAGGTCAC ATGCGTGGTG GTGGACGTGA 1450 GCCACGAAGA CCCTGAGGTC AAGTTCAACTGGTACGTGGA CGGCGTGGAG 1500 GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACAACAGCACGTA 1550 CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA1600 AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG 1650AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC 1700 CCTGCCCCCATCCCGGGAAG AGATGACCAA GAACCAGGTC AGCCTGACCT 1750 GCCTGGTCAA AGGCTTCTATCCCAGCGACA TCGCCGTGGA GTGGGAGAGC 1800 AATGGGCAGC CGGAGAACAA CTACAAGACCACGCCTCCCG TGCTGGACTC 1850 CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGACAAGAGCAGGT 1900 GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC1950 AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AATGA 1995 664 aminoacids Amino Acid Linear 16 Met Ile Leu Ala Asn Val Phe Phe Leu Phe PhePhe Leu Asp Glu 1 5 10 15 Thr Leu Arg Ser Leu Ala Ser Pro Ser Ser LeuGln Asp Pro Glu 20 25 30 Leu His Gly Trp Arg Pro Pro Val Asp Cys Val ArgAla Asn Glu 35 40 45 Leu Cys Ala Ala Glu Ser Asn Cys Ser Ser Arg Tyr ArgThr Leu 50 55 60 Arg Gln Cys Leu Ala Gly Arg Asp Arg Asn Thr Met Leu AlaAsn 65 70 75 Lys Glu Cys Gln Ala Ala Leu Glu Val Leu Gln Glu Ser Pro Leu80 85 90 Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu Leu Gln Cys 95100 105 Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu Thr Glu Gly Glu 110115 120 Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser Arg Leu 125130 135 Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr Gly Ala 140145 150 Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 155160 165 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser 170175 180 Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys 185190 195 Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg 200205 210 Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln 215220 225 Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser 230235 240 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg 245250 255 Gly Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp 260265 270 Phe His Ala Asn Cys Arg Ala Ser Tyr Gln Thr Val Thr Ser Cys 275280 285 Pro Ala Asp Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met 290295 300 Ile Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr 305310 315 Gly Ile Val Val Ser Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn 320325 330 Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp Phe Thr Glu Asn 335340 345 Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn Gly Thr Asp 350355 360 Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gln Ala Thr Gln Ala 365370 375 Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp 380385 390 Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Val 395400 405 Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met 410415 420 Cys Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly Pro Arg Asp 425430 435 Pro Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 440445 450 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 455460 465 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 470475 480 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 485490 495 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 500505 510 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 515520 525 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 530535 540 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 545550 555 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 560565 570 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 575580 585 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 590595 600 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 605610 615 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 620625 630 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 635640 645 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 650655 660 Ser Pro Gly Lys 664 1995 base pairs Nucleic Acid Single Linear17 ATGATCTTGG CAAACGCCTT CTGCCTCTTC TTCTTTTTAG ACGAAACCCT 50 CCGCTCTTTGGCCAGCCCTT CCTCCCTGCA GGGCTCTGAG CTCCACGGCT 100 GGCGCCCCCA AGTGGACTGTGTCCGGGCCA ATGAGCTGTG TGCGGCTGAA 150 TCCAACTGCA GCTCCAGGTA CCGCACCCTTCGGCAGTGCC TGGCAGGCCG 200 GGATCGCAAT ACCATGCTGG CCAATAAGGA GTGCCAGGCAGCCCTGGAGG 250 TCTTGCAGGA AAGCCCACTG TATGACTGCC GCTGCAAGCG GGGCATGAAG300 AAGGAGCTGC AGTGTCTGCA GATCTACTGG AGCATCCATC TGGGGCTGAC 350AGAGGGTGAG GAGTTCTATG AAGCTTCCCC CTATGAGCCT GTGACCTCGC 400 GCCTCTCGGACATCTTCAGG CTCGCTTCAA TCTTCTCAGG GACAGGGACA 450 GACCCGGCGG TCAGTACCAAAAGCAACCAC TGCCTGGATG CCGCCAAGGC 500 CTGCAACCTG AATGACAACT GCAAGAAGCTTCGCTCCTCT TATATCTCCA 550 TCTGCAACCG TGAGATCTCT CCCACCGAAC GCTGCAACCGCCGCAAGTGC 600 CACAAGGCTC TGCGCCAGTT CTTTGACCGT GTGCCCAGCG AGTATACCTA650 CCGCATGCTC TTCTGCTCCT GTCAGGACCA GGCATGTGCT GAGCGTCGCC 700GGCAAACCAT CCTGCCCAGT TGCTCCTATG AGGACAAGGA GAAGCCCAAC 750 TGCCTGGACCTGCGCAGCCT GTGTCGTACA GACCACCTGT GCCGGTCCCG 800 ACTGGCAGAT TTCCACGCCAACTGTCGAGC CTCCTACCGG ACAATCACCA 850 GCTGTCCTGC GGACAACTAC CAGGCATGTCTGGGCTCCTA TGCTGGCATG 900 ATTGGGTTTG ATATGACACC CAACTATGTG GACTCCAACCCCACGGGCAT 950 CGTGGTGTCT CCCTGGTGCA ATTGTCGTGG CAGTGGGAAC ATGGAAGAAG1000 AGTGTGAGAA GTTCCTCAGG GACTTCACGG AAAACCCATG CCTCCGGAAT 1050GCCATTCAGG CCTTTGGTAA TGGCACAGAT GTGAACATGT CTCCCAAAGG 1100 CCCCTCACTCCCAGCTACCC AGGCCCCTCG GGTGGAGAAG ACTCCTTCAC 1150 TGCCAGATGA CCTCAGTGACAGCACCAGCC TGGGGACCAG TGTCATCACC 1200 ACCTGCACAT CTATCCAGGA GCAAGGGCTGAAGGCCAACA ACTCCAAAGA 1250 GTTAAGCATG TGCTTCACAG AGCTCACGAC AAACATCAGTCCAGGGTCTA 1300 GAGACCCGGT GGACAAAACT CACACATGCC CACCGTGCCC AGCACCTGAA1350 CTCCTGGGGG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC 1400CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGA 1450 GCCACGAAGACCCTGAGGTC AAGTTCAACT GGTACGTGGA CGGCGTGGAG 1500 GTGCATAATG CCAAGACAAAGCCGCGGGAG GAGCAGTACA ACAGCACGTA 1550 CCGTGTGGTC AGCGTCCTCA CCGTCCTGCACCAGGACTGG CTGAATGGCA 1600 AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGCCCCCATCGAG 1650 AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC1700 CCTGCCCCCA TCCCGGGAAG AGATGACCAA GAACCAGGTC AGCCTGACCT 1750GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGC 1800 AATGGGCAGCCGGAGAACAA CTACAAGACC ACGCCTCCCG TGCTGGACTC 1850 CGACGGCTCC TTCTTCCTCTACAGCAAGCT CACCGTGGAC AAGAGCAGGT 1900 GGCAGCAGGG GAACGTCTTC TCATGCTCCGTGATGCATGA GGCTCTGCAC 1950 AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTAAATGA 1995 664 amino acids Amino Acid Linear 18 Met Ile Leu Ala Asn AlaPhe Cys Leu Phe Phe Phe Leu Asp Glu 1 5 10 15 Thr Leu Arg Ser Leu AlaSer Pro Ser Ser Leu Gln Gly Ser Glu 20 25 30 Leu His Gly Trp Arg Pro GlnVal Asp Cys Val Arg Ala Asn Glu 35 40 45 Leu Cys Ala Ala Glu Ser Asn CysSer Ser Arg Tyr Arg Thr Leu 50 55 60 Arg Gln Cys Leu Ala Gly Arg Asp ArgAsn Thr Met Leu Ala Asn 65 70 75 Lys Glu Cys Gln Ala Ala Leu Glu Val LeuGln Glu Ser Pro Leu 80 85 90 Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys LysGlu Leu Gln Cys 95 100 105 Leu Gln Ile Tyr Trp Ser Ile His Leu Gly LeuThr Glu Gly Glu 110 115 120 Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro ValThr Ser Arg Leu 125 130 135 Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe SerGly Thr Gly Thr 140 145 150 Asp Pro Ala Val Ser Thr Lys Ser Asn His CysLeu Asp Ala Ala 155 160 165 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys LysLeu Arg Ser Ser 170 175 180 Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser ProThr Glu Arg Cys 185 190 195 Asn Arg Arg Lys Cys His Lys Ala Leu Arg GlnPhe Phe Asp Arg 200 205 210 Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu PheCys Ser Cys Gln 215 220 225 Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln ThrIle Leu Pro Ser 230 235 240 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn CysLeu Asp Leu Arg 245 250 255 Ser Leu Cys Arg Thr Asp His Leu Cys Arg SerArg Leu Ala Asp 260 265 270 Phe His Ala Asn Cys Arg Ala Ser Tyr Arg ThrIle Thr Ser Cys 275 280 285 Pro Ala Asp Asn Tyr Gln Ala Cys Leu Gly SerTyr Ala Gly Met 290 295 300 Ile Gly Phe Asp Met Thr Pro Asn Tyr Val AspSer Asn Pro Thr 305 310 315 Gly Ile Val Val Ser Pro Trp Cys Asn Cys ArgGly Ser Gly Asn 320 325 330 Met Glu Glu Glu Cys Glu Lys Phe Leu Arg AspPhe Thr Glu Asn 335 340 345 Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe GlyAsn Gly Thr Asp 350 355 360 Val Asn Met Ser Pro Lys Gly Pro Ser Leu ProAla Thr Gln Ala 365 370 375 Pro Arg Val Glu Lys Thr Pro Ser Leu Pro AspAsp Leu Ser Asp 380 385 390 Ser Thr Ser Leu Gly Thr Ser Val Ile Thr ThrCys Thr Ser Ile 395 400 405 Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser LysGlu Leu Ser Met 410 415 420 Cys Phe Thr Glu Leu Thr Thr Asn Ile Ser ProGly Ser Arg Asp 425 430 435 Pro Val Asp Lys Thr His Thr Cys Pro Pro CysPro Ala Pro Glu 440 445 450 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe ProPro Lys Pro Lys 455 460 465 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val 470 475 480 Val Asp Val Ser His Glu Asp Pro Glu Val LysPhe Asn Trp Tyr 485 490 495 Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu 500 505 510 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val SerVal Leu Thr Val 515 520 525 Leu His Gln Asp Trp Leu Asn Gly Lys Glu TyrLys Cys Lys Val 530 535 540 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu LysThr Ile Ser Lys 545 550 555 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro 560 565 570 Ser Arg Glu Glu Met Thr Lys Asn Gln Val SerLeu Thr Cys Leu 575 580 585 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu Ser 590 595 600 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr ThrPro Pro Val Leu 605 610 615 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser LysLeu Thr Val Asp 620 625 630 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe SerCys Ser Val Met 635 640 645 His Glu Ala Leu His Asn His Tyr Thr Gln LysSer Leu Ser Leu 650 655 660 Ser Pro Gly Lys 664 670 base pairs NucleicAcid Single Linear 19 CTGCAAGAAG CTGCGCTCCT CCTACATCTC CATCTGCAACCGCGAGATCT 50 CGCCCACCGA GCGCTGCAAC CGCCGCAAGT GCCACAAGGC CCTGCGCCAG 100TTCTTCGACC GGGTGCCCAG CGAGTACACC TACCGCATGC TCTTCTGCTC 150 CTGCCAAGACCAGGCGTGCG CTGAGCGCCG CCGGCAAACC ATCCTGCCCA 200 GCTGCTCCTA TGAGGACAAGGAGAAGCCCA ACTGCCTGGA CCTGCGTGGC 250 GTGTGCCGGA CTGACCACCT GTGTCGGTCCCGGCTGGCCG ACTTCCATGC 300 CAATTGTCGA GCCTCCTACC AGACGGTCAC CAGCTGCCCTGCGGACAATT 350 ACCAGGCGTG TCTGGGCTCT TATGCTGGCA TGATTGGGTT TGACATGACA400 CCTAACTATG TGGACTCCAG CCCCACTGGC ATCGTGGTGT CCCCTGGTGC 450AGCTGTCGTG GCAGCGGGAA CATGGAGGAG GAGTGTGAGA AGTTCCTCAG 500 GACTTCACCGAGAACCCATG CCTCCGGAAC GCCATCCAGG CCTTTGGCAA 550 CGGCACGGAC GTGAACGTGTCCCCAAAAGG CCCTCGTTCC AGGCCACCCA 600 GGCCCCTCGG GTGGAGAAGA CGCCTTCTTTGCCAGATGAC CTCAGTGACA 650 GTACCAGCTT GGGGACCAGT 670

What is claimed is:
 1. A polypeptide comprising a sequence selected fromthe group consisting of: (a) a human NTNRα extracellular domain aminoacid sequence; (b) an allelic variant or mammalian homolog of (a); (c) asequence encoded by nucleic acid which hybridizes under stringentconditions to a nucleic acid encoding (a) or (b); (d) a sequence derivedfrom (a) or (b) by substitution, deletion, or addition of one or severalamino acids in the amino acid sequence of (a) or (b); and, (e) asequence encoding at least 16 contiguous amino acids from (a), (b) or(c).
 2. The polypeptide of claim 1, comprising the amino acid sequenceof NTNRα ECD from SEQ ID NO: 3 or SEQ D NO:
 6. 3. The polypeptide ofclaim 2, comprising the amino acid sequence of mature NTNRα from SEQ IDNO: 3 or SEQ ID NO:
 6. 4. The polypeptide of claim 3, which specificallybinds neurturin.
 5. The polypeptide of claim 4, which is conjugatedwith, or fused to, a molecule which increases the serum half-lifethereof.
 6. The polypeptide of claim 1 that is soluble NTNRα.
 7. Acomposition comprising the polypeptide of claim 1 and a physiologicallyacceptable carrier.
 8. The NTNRα of claim 1 that is chimeric NTNRα. 9.The chimeric NTNRα of claim 8, comprising a NTNRα amino acid sequencefused to an immunoglobulin sequence.
 10. The chimeric NTNRα of claim 8,comprising a NTNRα amino acid sequence fused to an epitope tag.
 11. Amethod for identifying a molecule which binds to the NTNRα, comprisingexposing the NTNRα to the molecule suspected of binding thereto anddetermining binding of the molecule to the NTNRα.
 12. The method ofclaim 11, wherein the NTNRα is soluble NTNRα.
 13. A method foridentifying a molecule which activates NTNRα, comprising exposing theNTNRα to a molecule suspected of being capable of activating NTNRα andmeasuring activation of NTNRα.
 14. A method for purifying a moleculewhich binds to NTNRα, comprising adsorbing the molecule to NTNRαimmobilized on a solid phase and recovering the molecule from theimmobilized NTNRα.
 15. The method of claim 14 wherein the NTNRα ischimeric NTNRα, comprising a fusion of a NTNRα extracellular domainsequence to an immunoglobulin constant domain sequence.
 16. An antibodythat specifically binds to NTNRα of claim
 1. 17. The antibody of claim16, which is a monoclonal antibody.
 18. A composition comprising theantibody of claim 17 and a physiologically acceptable carrier.
 19. Thecomposition of claim 18 further comprising a cytokine or a neurotrophicfactor.
 20. A method for activating NTNRα in a cell, comprising exposingthe NTNRα to an amount of an agonist antibody of claim 16 which iseffective for activating NTNRα.
 21. A method for modulating aphysiological response of a cell to NTN, comprising contacting the cellwith an amount of a NTNRα effective for modulating the response of thecell to NTN.
 22. A method for activating Ret on the surface of a cell,comprising administering to the cell a Ret-activating effective amountof soluble NTNRα or soluble GDNFRα.
 23. A method for determining thepresence of NTNRα, comprising exposing a test sample suspected ofcontaining the NTNRα to the antibody of claim 16 and determining bindingof said antibody to the test sample.
 25. An isolated nucleic acidmolecule comprising a sequence encoding a polypeptide of claim
 1. 26. Anisolated nucleic acid molecule comprising a sequence encoding apolypeptide of claim
 2. 27. An isolated nucleic acid molecule encodingthe polypeptide of claim
 8. 28. The isolated nucleic acid molecule ofclaim 25, further comprising a promoter operably linked to the nucleicacid molecule.
 29. An expression vector comprising the nucleic acidmolecule of claim 25 operably linked to control sequences recognized bya host cell transformed with the vector.
 30. A host cell comprising thevector of claim
 29. 31. A process of using a nucleic acid moleculeencoding NTNRα to effect production of NTNRα, comprising culturing thehost cell of claim 30 under conditions allowing expression of NTNRα. 32.The process of claim 31 further comprising recovering the NTNRα from thehost cell culture.
 33. A non-human, transgenic animal which containscells that express nucleic acid comprising the polypeptide of claim 1.34. The animal of claim 33 which is a mouse.
 35. A non-human, transgenicanimal which contains cells having an altered NTNRα gene.
 36. The animalof claim 35 which is a mouse.